Immunotherapy composition and use thereof

ABSTRACT

The present invention relates to the field of preventing or reducing incidence or severity of an allergic immune response, and compositions for preventing or reducing incidence or severity of an allergic immune response. For example, the present invention provides compositions comprising inactivated and/or killed cells of  Helicobacter pylori  or a cell lysate thereof, and methods and/or uses thereof for delaying or preventing or interrupting or slowing onset of one or more allergic conditions in a subject.

FIELD OF THE INVENTION

The present invention relates to the field of preventing or reducingincidence or severity of an allergic immune response, and compositionsfor preventing or reducing incidence or severity of an allergic immuneresponse.

BACKGROUND TO THE INVENTION

Allergic reactions are generally immune reactions that are initiated byIgE-dependent stimulation of tissue mast cells and related effectormolecules (e.g., basophils). Binding events between cell surface boundIgE molecules and antigen results in rapid release of biologicalresponse modifiers which bring about increased vascular permeability,vasodilation, smooth muscle contraction and local inflammation. Thissequence of events is termed immediate hypersensitivity and beginsrapidly, usually within minutes of exposure in a sensitised individual.In its most severe systemic form, anaphylaxis, such immediatehypersensitivity can bring about asphyxiation, produce cardiovascularcollapse, and even result in death. Individuals that are prone to strongimmediate hypersensitivity responses are referred to as “atopic”.Clinical manifestations of allergy or atopy include hay fever(rhinitis), asthma, urticaria (hives), skin irritation (e.g., eczemasuch as chronic eczema), anaphylaxis, and related conditions.

The prevalence of atopy has increased in the developed world since thebeginning of the 20^(th) century when allergy prevalence was estimatedto be less than 0.1% in Europe, UK and US (Schadewaldt H, 1980,Geschichte der Allergies in vier Dustri-Verlag; as cited by MatthiasWjst, 2009, Allergy, Asthma & Clinical Immunology. 5:8). About 30-40% ofthe world population is now affected by one or more allergic conditions.Asthma, rhinitis, and eczema are now prevalent in developed countries,with allergic disorders being the most common chronic diseases amongchildren in developed countries. For example, more than 25% of infantsin Australia today present with eczema, more than 20% of one-year oldsare food-sensitised, more than 25% of children have asthma, and morethan 40% of adults have a history of allergic rhinitis (Pawnkar R,Walter Canonica G, Holgate S T, Lockey R F, 2001, World AllergyOrganization (WAO) White Book on Allergy). Allergies also affect about20% of all individuals in the United States. Atopy is predicted toincrease to about 26 of the Australian population by 2050.

Although childhood asthma often improves during childhood, asthma andrhinitis persist throughout adulthood, with substantial increase inasthma associated mortality for those aged more than 60 years (Martin PE et al., 2011, J. Allergy Clin. Immunol. 127:1473-1479).

There is a significant economic burden associated with allergicconditions. For example, in 2007 the associated economic cost inAustralia was estimated to be $9.4 billion with an additional $21.3billion from lost wellbeing (e.g., disability and premature death). Inthe UK, the total annual expenditure for atopic eczema has beenestimated at £465 million (

521 m). In Germany, the total average costs for an atopic eczema patienthave been estimated to be about

4400. In the US, the direct and indirect costs of asthma to the USeconomy were projected to have reached US$20.7 billion in 2010, and thedirect cost of treating childhood asthma alone exceeds US$1,100 perpatient per annum. The cost of treating incidence of eczema alone inpatients aged 0 to 5 years is approximately US$360 per patient per annumwith an annual cost of over $400,000 in Australia,

5 million in Western Europe and US$3 million in the US.

Several studies have documented temporal changes in allergy patterns indeveloped countries, from a prevalence of allergic asthma and hay feverin children (Mullins R J, 2007, Med J Aust. 186: 618-621) towardincreasing eczema and food allergies during the last 10 years. In thissecond wave of the allergy epidemic, 10% of children have some form offood allergy (Osborne N J et al., 2011, J. Clin. Immunol. 127:668-676;Prescott S and Allen K J, 2011, Pediatr. Allergy Immunol. 22:155-160).This changing epidemiology for allergic disorders remains largelyunexplained.

As illustrated in panel (A) of FIG. 1 hereof, infants who have moderateto severe eczema are at higher risk of developing food allergies and/orallergic asthma later in life e.g., during childhood, and a significantproportion of these individuals will have atopic or respiratoryallergies as adults. This is the so-called “atopic march” or “allergicmarch” (Martin P E et al., 2011, J. Allergy Clin. Immunol.127:1473-1479). The current generation with food allergies appear topresent with symptoms earlier in life than previous generations havingrespiratory allergies, and appear less likely to outgrow their allergyduring early adulthood (Prescott S and Allen K J, 2011, Pediatr. AllergyImmunol. 22:155-160).

The so-called “hygiene hypothesis” attributes the increase in atopy indeveloped countries to an increase in the use of antibiotics to treatmicrobial infections in infancy and/or childhood (Strachan D P, 1989,BMJ, 299:1259-1260; Strachan D P, Harkins L S, Golding J, 1997, Clin.Exp. Allergy. 27:151-155; Renz H and Herz U, 2002, Eur. Respir. J.19:158-171). According to the hygiene hypothesis, changes in thebiodiversity of the microbial environment, human microbiome, and reducedexposure to microbes that regulate the host immune system causechildhood allergy leading to the atopic march e.g., because antibioticsreduce the incidence of microorganisms that are beneficial for abalanced immune system development in addition to reducing the incidenceof pathogens (Guarner F et al., 2006, Nat. clin. Pract. Gastroenterol.Hepatol. 3: 275-284).

Selection of an appropriate T-cell population occurs during the earlystages of immune responses in naive unsensitised hosts such as neonatesand new borns and infants having an undeveloped immune system. Ifselection favours priming the host immune system toward the induction ofallergen-specific TH1 cells, then IgG and IgA responses may ensue. TH1cells seem to play a role in defense against various microbial antigensincluding bacterial, viral and fungal infections, and uncontrolled TH1responses are involved in organ-specific autoimmunity e.g., inrheumatoid arthritis, multiple sclerosis, thyroiditis, Crohn's disease,systemic lupus erythematosus, experimental autoimmune uveoretinitis(Dubey et al., 1991, Eur. Cytokine Network, 2:147-152), experimentalautoimmune encephalitis (EAE) (Beraud et al., 1991, Cell Immunol.133:379-389), insulin-dependent diabetes mellitus (Hahn et al., 1987,Eur. J Immunol. 18:2037-2042), contact dermatitis (Kapsenberg et al.,Immunol Today, 12:392-395), and in some chronic inflammatory disorders.The principal inflammatory cytokine produced by TH1 cells is IFN-γ (See,for example, Romragnani, ed, TH1 and TH2 Cells in Health and Disease.Chem. Immunol., Karger, Basel, 63, pp. 158-170 and 187-203 (1996)).

On the other hand, the emergence of TH2 cells can lead to IgE productionand eosinophilia and ultimately atopic disease. See e.g., WO2005/030249.Allergy, asthma, eczema, psoriasis, allergic rhinitis, hay fever andatopic dermatitis are each associated with a profound immunologicalderegulation characterized by over production of TH2 cells (Romragnani,supra; van der Heijden et al., 1991; J Invest Derm. 97:389-394; Walkeret al., 1992, Am. Rev. Resp. Dis. 148:109-115; and Renz H and Herz U,2002, supra), and uncontrolled TH2 type responses are responsible fortriggering allergic disorders against environmental allergens andchemical allergens. TH2 type responses are also preferentially inducedin certain primary immune deficiencies such as hyper-IgE syndrome (DelPrete et al., 1989, J. Clin. Invest. 84:1830-1835) and Omenn's syndrome(Schandene et al., 1993, Eur. J. Immunol. 23:56-60).

TH2 effector functions may be negatively regulated by TH1 cells. Thehygiene hypothesis suggests that a reduced frequency of microbialinfections, less severe infection, and prevention of infection e.g., byfrequent use of antibiotics may prevent maturation of TH1 immunity, andgive rise to allergen-specific TH-2 immune responses followingsubsequent exposure to allergens (Renz H and Herz U, 2002, supra).

There is currently no cure, and only limited treatment, for severeatopy. Treatment options are generally restricted to use of steroids,anti-histamines, immune modulation drugs and administration ofadrenalin. At best these treatment regimens provide temporary relief andare generally not suitable for sustained use. Accordingly, there remainsan unmet need in the art for compositions and methods for prevention ofallergic disorders.

H. pylori is a gastric bacterial pathogen that chronically infects morethan half of the world's human population. Infection with H. pylori isusually acquired early in childhood and, if left untreated, can last fora life time with the majority of infected individuals remainingasymptomatic. On the other hand, H. pylori infection is the main causeof peptic ulcer disease, which is manifested in more than 10% ofinfected subjects (Kuipers et al., 1995. Aliment Pharmacol Ther, 9 Suppl2: 59-69). H. pylori infection is also associated with an increased riskof non-cardiac gastric adenocarcinoma which is one of the mostfrequently lethal malignancies, and with gastric mucosa-associatedlymphoid tissue (MALT) lymphoma (Suerbaum & Michetti, 2002, N Engl JMed, 347: 1175-1186; Atherton (2006), Annu Rev Pathol. 1:63-96), as wellas chronic urticaria (hives).

Epidemiological population studies suggest that prevalence of live H.pylori in the gastric mucosa is inversely-proportional to the incidenceof allergy in developed countries. See e.g., Zevit et al., (2011),Helicobacter, 17: 30-35; Shiotani et al., (2008), BMJ, 320: 412-7; Chen& Blaser (2007), Arch Intern Med, 167: 281-7; McCune et al., (2003), EurJ Gastroenterol Hepatol, 15: 637-40; Reibman et al., (2008), PLoS ONE,3: e4060; Konturek et al., (2008), Med Sci Monit, 14:CR453-8. However, anumber of other studies have suggested that the correlation betweenfalling H. pylori infection rates and raising allergy rates might not becorrect. See, e.g., Zevit et al., (2011) supra; Raj et al. (2009), JInfect Dis, 199:914-5. These conflicting reports suggest uncertainty asto whether or not reduced colonization of the gastric mucosa by H.pylori is directly involved in the atopic march.

SUMMARY OF THE INVENTION

1. General

In work leading to the present invention, the inventors sought toidentify and/or prepare composition(s) for improving tolerance of theimmune system of a mammalian subject to allergy e.g., by preventing ordelaying the development of atopy or the atopic march in a subject. Inparticular, the inventors sought to identify and/or preparecomposition(s) capable of preventing or reducing severity or incidenceof allergic immune response(s) to an allergen in a mammalian subject, orcapable of preventing or attenuating severity of allergic disease suchas airway hyper-responsiveness in a mammalian subject following exposureof the subject to an allergen. The inventors also sought to identifyand/or prepare composition(s) capable of preventing or interrupting orlimiting the atopic march and progression of an allergic disease such aseczema in children e.g., neonates and juveniles to food allergy and/orsevere asthma later in life for example during adolescence and/oradulthood.

The inventors reasoned that an optimally-balanced immune system developsin the early post-natal period and, as a consequence, administration ofa medicament to prevent the atopic march in a subject and development ofallergy in adolescents and adults is optimally deliverable to neonatesor during early childhood.

As exemplified herein, the present inventors have shown that an oralcomposition comprising inactivated and/or killed H. pylori administeredto neonates or adults in a murine model of allergy reduced the incidenceor severity of an allergic response to antigenic challenge e.g., asdetermined by measurement of airway or lung resistance. Thus,administration of an inactivated and/or killed H. pylori e.g., whereinthe inactivated H pylori does not have the same capacity of a live H.pylori to colonize the mucosa of a mammal to which it is administered orwherein the inactivated or killed H. pylori is incapable of colonizingthe mucosa of a mammal to which it is administered, or a H. pylori celllysate, appears to interrupt or slow or arrest or prevent atopic marchor further atopic march in the subject e.g., by delaying or preventingor interrupting or slowing the onset of one or more allergic conditionssuch as allergic eczema, urticaria, hives, rhinitis, wheezing, airwayresistance, airway restriction, or airway hyper-responsiveness orhyper-reactivity, food allergy, asthma etc.

The present invention therefore provides for a general reduction inhyper-responsiveness of an individual to one or more allergens therebydelaying or preventing or interrupting or slowing the onset of one ormore allergic conditions. The reduced hypersensitivity may bedemonstrated by reduced sensitivity of a subject to a specific allergene.g., an accepted model allergen of hypersensitivity e.g., ovalbuminand/or ragweed administered as a challenge to murine animals e.g.,BALB/c or C57/BL/6 or SJL/J mice, in an aerosolized form or by gavage.See e.g., Renz et al., J. Allergy Clin. Immunol. 89:1127-1138 (1992);Renz et al., J. Immunol. 151:1907-1917 (1993); Saloga et al., J. Clin.Invest. 91:133-140 (1993); Larsen et al., J. Clin. Invest. 89:747-752(1992); Oshiba et al., J. Clin, Invest. 97: 1938-1408 (1996).

For example, by administering inactivated and/or killed H. pylori e.g.,isolated inactivated and/or killed H. pylori, to a subject that isasymptomatic for eczema, or asymptomatic for allergy e.g., characterizedby rhinitis or wheezing or airway resistance or restriction or airwayhyper-responsiveness, or asymptomatic for asthma, a subsequent onset ofeczema and/or allergy and/or asthma may be prevented. In one specificexample, inactivated and/or killed H. pylori is administered to ajuvenile subject such as a neonate or infant to prevent eczema in theinfant or a subsequent onset of allergy or asthma in later life e.g., inadolescence or adulthood. In another example, inactivated and/or orkilled H. pylori e.g., isolated inactivated and/or killed H. pylori, isadministered to an adolescent or adult subject to prevent eczema in thesubject or a subsequent onset of allergy or asthma, such as in laterlife. This is in a background in which allergic eczema, allergy orasthma is inducible at any stage of life by exposure of a subject to oneor more challenge allergens, including one or more environmentalallergens e.g., pollen allergen, dust mite allergen, animal allergen,chemical allergen etc.

Alternatively, by administering inactivated and/or killed H. pylorie.g., isolated inactivated and/or killed H. pylori, to a subject thathas suffered previously from one or more incidences of allergic eczema,allergy e.g., characterized by rhinitis or wheezing or airway resistanceor restriction, or asthma, a subsequent attack may be prevented or theseverity of a subsequent attack may be reduced. In one specific example,inactivated and/or killed H. pylori e.g., isolated inactivated and/orkilled H. pylori is administered to a juvenile subject that has sufferedfrom allergic eczema to prevent a subsequent attack or reduce severityof a subsequent attack, optionally to prevent or slow further atopicmarch in the subject. In another example, inactivated and/or killed H.pylori e.g., isolated inactivated and/or killed H. pylori isadministered to an adolescent or adult subject that has sufferedpreviously from allergic eczema and/or allergy and/or asthma, to preventa subsequent attack or reduce severity of a subsequent attack,optionally to prevent or slow further atopic march in the subject.

In an epidemiological context, the administration of inactivated and/orkilled H. pylori e.g., isolated inactivated and/or killed H. pylori to asubject reduces the incidence of allergic immune responses in thepopulation, and especially reduces the incidence of allergic immuneresponses in adolescent and/or adult members of the population treatedwhen they were juveniles.

The demonstration that inactivated and/or killed H. pylori bacteriaprotect subjects in a mouse model of allergic airway disease providesthe significant advantage of avoiding health risks associated with theuse of live H. pylori cells, such as induction of peptic ulcers and/orgastric cancer. In other words, inactivated and/or killed H. pylori or alysate of H. pylori offers a safe and controlled approach forpositively-influencing the developing immune system, and preventing orreducing an allergic response to an allergen. Similarly, inactivatedand/or killed H. pylori or a lysate of H. pylori offers a safe andcontrolled approach to delaying or preventing the atopic march bytargeting events in early in life e.g., in children such as neonatesand/or juveniles.

The present invention thus provides for administration, for examplerepeated administration, of inactivated and/or killed H. pylori bacteriaand/or a lysate thereof e.g., to children or infants such as at 0 to 5years of age, to thereby promote balanced immune development forreducing the severity or incidence of allergy e.g., as allergic eczemaand/or a life-long food allergy and/or allergic asthma. The inactivatedand/or killed H. pylori bacteria and/or a lysate thereof is also usefulfor modulating the immune system of a mammalian subject and/or forimproving the immune system's tolerance to allergy.

As disclosed herein, the inactivated and/or killed H. pylori bacteriaand/or a lysate thereof are formulated and/or used as a food ingredientor a food product such as medical food e.g., diary or non-dairy and/ordietary supplement(s) and/or as tablet(s) and/or as capsules. Suchformulations are preferably mucosal compositions for improving immunesystem's tolerance to allergens and/or preventing or reducing allergysymptoms for example in adults and/or adolescents. The formulations arepreferably for repeated administration, e.g., daily, to children and/orinfants, e.g., aged 0 to 5 years, suffering from eczema and/or foodallergy or susceptible to development of eczema or food allergy. In thisrespect, a subject may be susceptible to development of allergy at 0-5years or 0-4 years or 0-3 year or 0-2 years or 0.5-5 years or 0.5-4years or 0.5-3 years or 0.5-2 years or 0.5-1 years or 1-2 years or 1-3years or 1-4 years or 1-5 years or 2-3 years or 2-4 years or 2-5 yearsor 3-4 years or 3-5 years of age. For example, to prevent or limit theatopic march in a subject, such as progression to food allergy and/orallergic asthma later in life, the subject is administered a pluralityof doses of a formulation comprising the inactivated H. pylori or cellextract or lysate thereof, wherein the first does is administered at atime infra where the subject is susceptible to development of allergy.For example, the subject may be taking antibiotic therapy or prescribedantibiotic therapy, especially in the case of an infant or child that issusceptible to development of allergy.

Without being bound by theory or specific mode of action, the inventorspostulated that the inactivated and/or killed H. pylori of the presentinvention retain and/or form a cell structure scaffold and/or aconglomerate or aggregate of cell structure scaffold. Without beingbound by theory or specific mode of action, the inventors alsopostulated that this scaffold and/or conglomerate or aggregate may beimportant for facilitating immune modulation in a subject towards abalanced immune response to an allergen e.g., balanced Th1/Th2 immuneresponse in a subject and/or to interrupt or slow or arrest or preventatopic march or further atopic march in the subject e.g., by delaying orpreventing or interrupting or slowing the onset of one or more allergicconditions described herein.

SPECIFIC EXAMPLES OF THE INVENTION

The scope of the invention will be apparent from the claims as filedwith the application that follow the examples. The claims as filed withthe application are hereby incorporated into the description. The scopeof the invention will also be apparent from the following description ofspecific embodiments and/or detailed description of preferredembodiments.

Accordingly, in one example, the invention provides a compositioncomprising an H. pylori cell, a cell lysate thereof or combinationthereof and a pharmaceutically accepted carrier, wherein said H. pyloricell is inactivated e.g., by virtue of having reduced capacity tocolonize the mucosa of a mammal relative to a live H. pylori cell suchas a live H. pylori cell having the same genotype as the inactivatedcell, or by virtue of being incapable of colonizing the mucosa of saidmammal, and preferably wherein the H. pylori is killed e.g., by heattreatment.

In some embodiments, the composition of the present invention consistsessentially of an H. pylori cell and/or a cell lysate thereof togetherwith a pharmaceutically acceptable carrier, wherein said H. pylori cellis inactivated e.g., by virtue of having reduced capacity to colonizethe mucosa of a mammal relative to a live H. pylori cell for examplehaving the same genotype as the inactivated cell, or by virtue of beingincapable of colonizing the mucosa of said mammal, and preferablywherein the H. pylori is killed e.g., by heat treatment.

It will be appreciated by those skilled in the art that any H. pyloristrain is used; however, in some examples the H. pylori strain is cagAminus (cagA⁻). In some examples, the H. pylori strain is cagA⁻ and isalso positive for the toxigenic s1 and m1 alleles of the vacA gene.

In some examples, the present invention provides strains of H. pylorihaving the characteristics of a strain selected from the groupconsisting of OND737, as deposited in the National Measurement Instituteunder Accession No. V09/009101; OND738, as deposited in the NationalMeasurement Institute under Accession No. V09/009102; OND739, asdeposited in the National Measurement Institute under Accession No.V09/009103; OND248, as deposited in the National Measurement Instituteunder Accession No. V10/014059; OND256 as deposited in the NationalMeasurement Institute under Accession No. V10/014060; OND740, asdeposited in the National Measurement Institute under Accession No.V09/009104; OND79, as deposited in the National Measurement Instituteunder Accession No. V13/023374, and/or OND86, as deposited in theNational Measurement Institute under Accession No. V14/013016, or apassaged strain, a mutant or a derivative thereof.

In some examples, the H. pylori strain of the present invention has beenpassaged through an animal host such as a human host. For example, theH. pylori strain of the present invention is derived from the H. pyloristrain OND79 after passage of the OND79 strain in a human subject e.g.,following infection and/or colonization of the gastric mucosa of a humansubject with H. pylori OND79 strain. In one such example, the H. pyloristrain of the present invention is OND86.

While the H. pylori strain used in the present invention is typically anon-genetically modified bacterium, in some examples the H. pyloristrain is genetically modified to comprise one or more nucleic acidmolecule(s) encoding at least one heterologous antigen or a functionalfragment thereof.

In some examples the nucleic acid molecule resides extra-chromosomallyon, for example, a plasmid vector such as a shuttle vector. Preferably,the plasmid vector would comprise (a) a nucleic acid sequence encodingthe heterologous antigen and (b) a control or regulatory sequenceoperatively linked thereto which is capable of controlling theexpression of the nucleic acid when the vector is transformed into a H.pylori strain. In other examples, the nucleic acid molecule inserts intothe H. pylori chromosome upon transformation into the H. pylori.

Suitable antigens will be known to the person skilled in the art.Preferably the antigen is an environmental antigen, and may be usedeither singly or as a combination of two or more such antigens.

In some examples, the composition of the present invention will comprisean adjuvant. The adjuvant may be any adjuvant known in the art; however,preferably, the adjuvant is selected from the group consisting of alum,petiussis toxin, lacto fucopentaose III, phosphopolymer, completeFreund's adjuvant, monophosphoryl lipid A, 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL), aluminium salt, CpG-containingoligonucleotides, immunostimulatory DNA sequences, saponin, MONTANIDE®1SA 720, SAF, ISCOM.S, MF-59®, SBAS-3, SBAS-4, Detox, RC-529, aminoalkylglucosaminide 4-phosphate, and LbeiF4A or combinations thereof.Alternatively, in other examples, the mucosal composition of the presentinvention does not comprise an adjuvant and/or is administered in theabsence of an adjuvant.

The invention is useful in preventing and/or treating allergy in amammal at risk of developing an allergy or having an allergy. In someexamples, the allergy is selected from the group consisting of contactdermatitis, chronic inflammatory disorders, allergic atopic disorders,allergic asthma, atopic dermatitis, hyper-IgE syndrome, Omenn'ssyndrome, psoriasis, hay fever, allergic rhinitis, urticaria, eczema andfood allergies.

Accordingly, in a further example the present invention provides acomposition for use in preventing or treating allergy in a mammalcomprising an H. pylori cell such as an isolated H. pylori cell, a celllysate thereof or combination thereof and a pharmaceutically acceptedcarrier, wherein said H. pylori cell is either killed or incapable ofcolonizing the mucosa of said mammal. Optionally, the cell lysate is awhole cell lysate (WCL) of the inactivated H. pylori cell.

In a further example, the present invention provides a compositioncomprising an H. pylori cell such as an isolated H. pylori cell, or acell lysate thereof or combination thereof and a pharmaceuticallyacceptable carrier, wherein said H. pylori cell is inactivated e.g., byvirtue of having reduced capacity to colonize the mucosa of a mammalrelative to a live H. pylori cell for example having the same genotypeas the inactivated cell or by virtue of being incapable of colonizingthe mucosa of a mammal. Preferably, the composition is for mucosaldelivery. Optionally, the cell lysate is a whole cell lysate (WCL) ofthe inactivated H. pylori cell.

In another example, the present invention provides a compositioncomprising inactivated and/or killed H. pylori cells, such as isolatedinactivated and/or killed H. pylori cells, or a cell lysate thereof,wherein said composition is formulated to be administered mucosally to asubject for interrupting or slowing or arresting or preventing an atopicmarch or progression of an atopic march in the subject. For example, thecell lysate is a WCL. In one such example, interrupting or slowing orarresting or preventing an atopic march or progression of an atopicmarch in the subject comprises delaying or preventing or interrupting orslowing the onset of one or more allergic conditions in the subject.

For example, an allergic condition may comprise allergic eczema,urticaria, hives, rhinitis, wheezing, airway resistance, airwayrestriction, lung inflammation, food allergy, or asthma. Preferably, anallergic condition comprises airway resistance or airwayhyperresponsiveness or hyperreactivity in response to an allergen andwherein the composition is for reducing said airway resistance.Alternatively, or in addition, an allergic condition comprises lunginflammation in response to an allergen and wherein the composition isfor reducing said lung inflammation e.g., as characterized by a reducedlevel of cell infiltrate in lung. Alternatively, or in addition, anallergic condition is characterized by an elevated serum level ofallergen-specific IgE antibody and/or an elevated level of one or moreinflammatory cytokines in bronchioalveolar lavage (BAL) and/or anelevated level of cell infiltrate in lung. For example, the compositionreduces a serum level of allergen-specific IgE antibody and/or a levelof one or more inflammatory cytokines in bronchioalveolar lavage (BAL)and/or a level of cell infiltrate in lung relative to a level thereof ina subject exposed to an allergen and not administered said composition.Alternatively, the composition prevents or delays an increase in a serumlevel of allergen-specific IgE antibody and/or prevents or delays anincrease in a level of one or more inflammatory cytokines inbronchioalveolar lavage (BAL) and/or prevents or delays an increase in alevel of cell infiltrate in lung in a subject exposed to an allergen.

Preferably, the composition as described according to any example hereofcomprises H pylori cells or strains which have reduced capability incolonizing the mucosa of a subject relative to live H pylori cells orstrains or are incapable of colonizing the mucosa of a subject.Alternatively, or in addition, the composition according to any exampledescribed hereof comprises H pylori cells or strains which areinactivated e.g., by irradiation such as gamma irradiation and/orultraviolet irradiation and/or heat treatment and/or chemical meansand/or by exposure to acid and/or by exposure to a base and/or byphysical means such as pressure and/or by lyophilisation and/or byfreeze-thawing. Alternatively, or in addition, the composition accordingto any example hereof comprises H. pylori cells or strains which arekilled e.g., by heat treatment such that the cells are renderedirreversibly metabolically inactive. In another example, the compositionaccording to any example hereof comprises H pylori cells or strains thathave been subjected to a process for inactivating H. pylori cells and aprocess for killing the H. pylori cells. In one particular example, theinactivated H. pylori cells or strains described according to anyexample hereof are killed.

Alternatively, or in addition, the composition described according toany example hereof comprises a lysate e.g., WCL of H. pylori cellswherein the cells have been subjected to a process for inactivating H.pylori cells and/or a process for killing the H. pylori cells.

For example, inactivated H. pylori as described according to any examplehereof is prepared by exposing live H. pylori cells or strains toirradiation such as gamma irradiation and/or ultraviolet irradiationand/or by exposure to visible light such as wavelengths ranging fromabout 375 nm to about 500 nm or in a range from about 400 nm to about420 nm e.g., 405 nm violet light. In one example, inactivated H. pylorias described according to any example hereof is prepared by a processcomprising exposing live H. pylori cells or strains to ultraviolet C(UVC) irradiation such as wavelength in a range from about 100 nm toabout 280 nm such as about 257.3 nm and/or to ultraviolet B (UVB)irradiation such as wavelength in a range from about 280 nm to about 315nm and/or to ultraviolet A (UVA) irradiation such as wavelength in arange from about 315 nm to about 400 nm. Preferably, the live H. pyloriis exposed to UVC light in a range from about 100 nm to about 280 nmsuch as about 257.3 nm and/or the live H. pylori is exposed to about 405nm violet light.

Alternatively, or in addition, inactivated H. pylori as describedaccording to any example hereof is prepared by exposing live H. pyloricells or strains to one or more chemical agents such as formaldehydeand/or β-propiolactone and/or ethyleneimine and/or binary ethyleneimineand/or thimerosal and/or polyethyleneimine functionalized zinc oxidenanoparticles, or derivatives thereof. For example, live H. pylori cellsor strains may be inactivated by exposure to formaldehyde at aconcentration from about 0.01% to about 1% (w/w) or from about 0.01% toabout 0.1% (w/w) or between about 0.025% and about 0.1% (w/w).

Alternatively, or in addition, inactivated H. pylori as describedaccording to any example hereof is prepared by exposing live H. pyloricells or strains to heat treatment such as at temperatures in the rangebetween about 40° C. to about 70° C. or more. Alternatively, or inaddition, inactivated H. pylori as described according to any examplehereof is prepared by exposing live H. pylori cells or strains to one ormore acid(s) or to a low pH environment such as pH 3.0 or lower and/orto one or more base(s) or to high pH environment such as pH 9.0 orhigher.

Alternatively, or in addition, inactivated H. pylori as describedaccording to any example hereof is prepared by exposing live H. pyloricells or strains to one or more reducing agent(s) such as sodiumbisulfite and/or one or more oxidative agents such as hydrogen peroxide.

Alternatively, or in addition, inactivated H. pylori as describedaccording to any example hereof is prepared by exposing live H. pyloricells or strains to bile salts.

Alternatively, or in addition, inactivated H. pylori as describedaccording to any example hereof is prepared by mutagenesis of live Hpylori cells or strains.

Alternatively, or in addition, inactivated H. pylori as describedaccording to any example hereof is prepared by lyophilizing orfreeze-drying live H. pylori cells or strains. Alternatively, or inaddition, inactivated H pylori as described according to any examplehereof is prepared by performing one or cycles of freezing and thawinglive H. pylori cells or strains.

For example, killed H. pylori as described according to any examplehereof is prepared by exposing live and/or inactivated H. pylori cellsor strains to heat treatment such as by exposure to temperature of about60° C. or more for at least about 60 seconds, preferably at atemperature of about 60° C. or about 70° C. or about 80° C. or about 90°C. or about 100° C. or about 110° C. or about 120° C. or about 130° C.or about 140° C. or about 150° C., said temperature exposure being for aperiod of at least 2 minutes or at least 3 minutes or at least 4 minutesor at least 5 minutes or at least 6 minutes or at least 7 minutes or atleast 8 minutes or at least 9 minutes or at least 10 minutes or at least20 minutes or at least 30 minutes or at least 40 minutes or at least 50minutes or at least 1 hour or at least 2 hours or at least 3 hours or atleast 4 hours or at least 5 hours or at least 6 hours or at least 7hours or at least 8 hours or at least 9 hours or at least 10 hours or atleast 11 hours or at least 12 hours or at least 13 hours or at least 14hours or at least 15 hours or at least 16 hours or at least 17 hours orat least 18 hours or at least 19 hours or at least 20 hours or at least21 hours or at least 22 hours or at least 23 hours or at least 1 day orat least 2 days or at least 3 days or at least 5 days or at least 5 daysor at least 6 days or at least 7 days. In one preferred example, liveand/or inactivated H. pylori is killed by exposure to a single suchelevated temperature or by exposure to at least two different elevatedtemperatures such as by exposure to a first temperature of about 70° C.followed exposure to a second temperature of about 90° C. or about 95°C. In one such preferred example, the live and/or inactivated H pyloriis killed by exposure to temperature of about 70° C. for about 10minutes followed by exposure to temperature of about 90° C. or about 95°C. for about 5 minutes.

Alternatively, or in addition, killed H. pylori as described accordingto any example hereof is prepared by exposing live and/or inactivated H.pylori cells or strains to elevated temperatures in the presence ofsteam and elevated pressure, such as by autoclaving live and/orinactivated H. pylori cells or strains. For example, live and/orinactivated H. pylori is killed by autoclaving the bacterial cells orstrains for about 15 minutes at about 121° C. and about 15 psi, or forabout 3 minutes at about at 132° C. and about 30 psi.

Alternatively, or in addition, killed H. pylori as described accordingto any example hereof is prepared by exposing live and/or inactivated H.pylori cells or strains to one or more bactericidal agent(s). Forexample, live and/or inactivated H. pylori can be subjected to treatmentwith one or more antibiotics selected from rifampin, amoxicillin,clarithromycin, rifamycin, rifaximin, the rifamycin derivative3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin syn.KRM-1648 and/or the rifamycin derivative3′-hydroxy-5′-(4-propyl-1-piperazinyl)benzoxazinorifamycin syn.KRM-1657.

Alternatively, or in addition, killed H. pylori as described accordingto any example hereof is prepared by exposing live and/or inactivated H.pylori cells or strains to irradiation such as gamma irradiation and/orultraviolet irradiation and/or by exposure to visible light such aswavelengths ranging from about 375 nm to about 500 nm or in a range fromabout 400 nm to about 420 nm. For example, killed H. pylori is preparedby a process comprising exposing live and/or inactivated H. pylori cellsor strains to ultraviolet C (UVC) irradiation such as wavelength in arange from about 100 nm to about 280 nm such as about 257.3 nm and/or toultraviolet B (UVB) irradiation such as wavelength in a range from about280 nm to about 315 nm and/or to ultraviolet A (UVA) irradiation such aswavelength in a range from about 315 nm to about 400 nm. Preferably, thelive and/or inactivated H. pylori is exposed to UVC light in a rangefrom about 100 nm to about 280 nm such as about 257.3 nm and/or the liveand/or inactivated H. pylori is exposed to about 405 nm violet light.

Alternatively, or in addition, killed H. pylori as described accordingto any example hereof is prepared by sonication e.g., at ultrasonicfrequencies such as about 20 kHz or more.

Alternatively, or in addition, killed H. pylori as described accordingto any example hereof is prepared by mutagenesis of live and/orinactivated H. pylori cells or strains.

Preferably, the killed H. pylori as described according to any examplehereof is prepared by first by exposing live H. pylori cells or strainsto irradiation such as gamma irradiation and/or ultraviolet irradiationsuch as UVC light and/or by exposure to visible light such aswavelengths ranging from about 375 nm to about 500 nm or in a range fromabout 400 nm to about 420 nm, to thereby inactivate H. pylori and thenexposing the inactivated H. pylori cells or strains to heat treatment asdescribed according to any example hereof to thereby kill theinactivated H. pylori or render the inactivated H. pylori irreversiblymetabolically inactive.

For example, the inactivated H. pylori is exposed to temperature ofabout 60° C. or more for at least about 60 seconds, preferably at atemperature of about 60° C. or about 70° C. or about 80° C. or about 90°C. or about 100° C. or about 110° C. or about 120° C. or about 130° C.or about 140° C. or about 150° C., said temperature exposure being for aperiod of at least 2 minutes or at least 3 minutes or at least 4 minutesor at least 5 minutes or at least 6 minutes or at least 7 minutes or atleast 8 minutes or at least 9 minutes or at least 10 minutes or at least20 minutes or at least 30 minutes or at least 40 minutes or at least 50minutes or at least 1 hour or at least 2 hours or at least 3 hours or atleast 4 hours or at least 5 hours or at least 6 hours or at least 7hours or at least 8 hours or at least 9 hours or at least 10 hours or atleast 11 hours or at least 12 hours or at least 13 hours or at least 14hours or at least 15 hours or at least 16 hours or at least 17 hours orat least 18 hours or at least 19 hours or at least 20 hours or at least21 hours or at least 22 hours or at least 23 hours or at least 1 day orat least 2 days or at least 3 days or at least 5 days or at least 5 daysor at least 6 days or at least 7 days. In one such example, theinactivated H. pylori is exposed to a single such elevated temperatureor to at least two different elevated temperatures such as by exposureto a first temperature of about 70° C. e.g., for about 10 minutes,followed by exposure to a second temperature of about 90° C. or about95° C. e.g., for about 5 minutes.

In one preferred example, the killed H. pylori as described according toany example hereof is prepared by first by exposing live H. pylori cellsor strains to ultraviolet irradiation such as UVC light e.g., at aboutas 257.3 nm to thereby inactivate H. pylori and then exposing theinactivated H. pylori cells or strains to heat treatment as describedaccording to any example hereof to thereby kill the inactivated H.pylori or render the inactivated H. pylori irreversibly metabolicallyinactive.

Accordingly, in one preferred example, the composition according to anyexample hereof comprises H. pylori that has been subjected to a processfor inactivating H. pylori by irradiation and a process for the killingthe inactivated H. pylori by heat treatment.

Alternatively, or in addition, H. pylori as described according to anyexample hereof is inactivated and/or killed by exposing live orinactivated H. pylori to anaerobic conditions e.g., by changing theatmosphere in which H. pylori is cultured from microaerobic to anaerobicenvironment for example to mimic the in vivo atmospheric conditionsduring the washout of H. pylori from the stomach to the lower gut (e.g.,small and/or large intestine). For example, live (such as freshly grown)H. pylori is inactivated by exposing (e.g., by growing or incubating)the bacterial cells to anaerobic conditions for about 1 day to about 5days or more, including for at least about 24 hours, or for at leastabout 48 hours or at least about 72 hours or at least about 96 hours orat least about 120 hours. In one such example, the live H. pylori cellsare inactivated by exposing the cells to anaerobic conditions and byheat treatment of the cells.

In another example, live or inactivated H. pylori as described accordingto any example hereof is killed by exposing (e.g., by incubation) thelive or inactivated bacterial cells to anaerobic conditions for about 1day to about 5 days or more, including for at least about 24 hours, orfor at least about 48 hours or at least about 73 hours or at least about96 hours or at least about 120 hours.

In one preferred example, the composition according to any examplehereof comprises H. pylori that has been subjected to a process forinactivating H. pylori by exposing (e.g., by growing or incubating) thebacterial cells to anaerobic conditions for about 1 day to about 5 daysor more, including for at least about 24 hours, or for at least about 48hours or at least about 73 hours or at least about 96 hours or at leastabout 120 hours, and a process for the killing the inactivated H. pyloriby heat treatment of the cells.

In a further example, the composition according to any example describedhereof comprises a pharmaceutically acceptable carrier. In one preferredexample, the composition does not include an adjuvant.

In a further example, the composition according to any example describedhereof is an oral formulation formulated for ingestion. Alternatively,the composition according to any example described hereof is formulatedfor inhalation. For example, the composition according to any exampledescribed hereof is formulated as a foodstuff or dietary supplement. Inone such example, the composition comprises or formulated as an infantformula and/or a protein supplement. In one example, the composition isa dairy food product or a non-dairy food product. In one example, thecomposition is formulated as a tablet e.g., for ingestion.Alternatively, the composition is in a powder form e.g., for ingestionand/or inhalation. Alternatively, the composition is in liquid form. Inone example, the composition does not include an adjuvant.

In one example, the composition according to any example describedhereof is formulated for administration (e.g., by consumption) toinfants, such as to infants who do not have developed lymphoidstructures. For example, the composition according to any exampledescribed hereof is formulated for administration to infants agedbetween 0 to about 5 years, or between 0 to about 4 years, or between 0to about 3 years, or between 0 to about 2 years, or between 0 to about 1year. In one example the composition according to any example describedhereof is formulated for administration (e.g., by consumption) toinfants aged between 0 to about 2 years. In another example, thecomposition is formulated for administration to infants of an agebetween about 4 months and about 12 months or between about 4 months andabout 18 months or about 4 months and about 24 months. In anotherexample, the composition is formulated for administration (e.g., byconsumption) to infants less than about 6 months of age.

In another example, the composition according to any example describedhereof is formulated for administration (e.g., by consumption) tochildren older than about 5 years of age and/or to adolescents and/or toadults.

In another example, the composition according to any example describedhereof is formulated for repeated administration, or is administeredrepeatedly, for example, once per week, or twice per week, or threetimes per week, or 4 times per week, or 5 times per week, or 6 times perweek, or 7 times per week, or more than 7 times per week, or more thantwice per day.

In one example, the composition according to any example describedhereof is formulated or administered as a multi-dosage unit composition.For example, each dosage of the composition comprises an amount of theH. pylori bacteria or a lysate thereof in a range corresponding tobetween about 10² cells to about 10¹⁴ cells, or about 10³ cells to about10¹³ cells, or about 10⁴ cells to about 10¹³ cells, or about 10⁵ cellsto about 10¹³ cells, or about 10⁶ cells to about 10¹³ cells, or about10⁶ cells to about 10¹² cells, or about 10⁷ cells to about 10¹¹ cells,or about 10⁸ cells to about 10¹⁰ cells, or about 10⁹ cells to about 10¹⁰cells. For example, each dosage of the composition comprises an amountof the H. pylori bacteria or a lysate thereof corresponding to about 10⁸cells, or about 10⁹ cells, or about 10¹⁰ cells. In one example, thecomposition according to any example described hereof is formulated foradministration daily, or is administered daily, wherein a daily dosageof said composition comprises an amount of the H. pylori bacteria or alysate thereof in a range corresponding to between about 10² cells toabout 10¹⁴ cells, or about 10³ cells to about 10¹³ cells, or about 10⁴cells to about 10¹³ cells, or about 10⁵ cells to about 10¹³ cells, orabout 10⁶ cells to about 10¹³ cells, or about 10⁶ cells to about 10¹²cells, or about 10⁷ cells to about 10¹¹ cells, or about 10⁸ cells toabout 10¹⁰ cells, or about 10⁹ cells to about 10¹⁰ cells. For example,each daily dosage of the composition comprises an amount of the H.pylori bacteria or a lysate thereof corresponding to about 10⁸ cells, orabout 10⁹ cells, or about 10¹⁰ cells.

In one example, the composition according to any example describedhereof is formulated for administration, or is administered, over aperiod of at least about 2 weeks or at least about 4 weeks or at leastabout 6 weeks or at least about 8 weeks or at least about 10 weeks or atleast about 11 weeks or at least about 12 weeks or at least about 13weeks at least about 14 weeks or at least about 15 weeks or at leastabout 16 weeks or at least about 17 weeks or at least about 18 weeks orat least about 19 weeks or at least about 20 weeks or at least about 21weeks or at least about 22 weeks or at least about 23 weeks or at leastabout 24 weeks or at least about 25 weeks, or at least about 6 months,or at least about one year or more than one year. Preferably, thecomposition according to any example described hereof is formulated foradministration, or is administered, over a period of at least about 13weeks or at least about 3 months.

In one example, the composition according to any example describedhereof is formulated for administration, or is administered, in absenceof an adjuvant and/or wherein said composition does not comprise anadjuvant.

In another example, the composition or a dosage (e.g., daily dosage) ofthe composition according to any example described hereof promotes abalanced development of an immune system in a juvenile subject. Inanother example, the composition or a dosage (e.g., daily dosage) of thecomposition according to any example described hereof promotesacquisition of adaptive immunity and/or innate immunity in a subject. Inanother example, the composition or a dosage (e.g., daily dosage) of thecomposition according to any example described hereof promotes orenhances CD1d receptor activation and/or CD4-negative and CD8-negativenatural killer (NK) cells in a subject.

In another example, the composition or a dosage (e.g., daily dosage) ofthe composition according to any example described hereof promotes orenhances γδ T-cell activation. In another example, the composition or adosage (e.g., daily dosage) of the composition according to any exampledescribed hereof promotes or enhances mucosal immunity involving immunerecognition and presentation to antigen-presenting cells (APCs). Inanother example, the composition or a dosage (e.g., daily dosage) of thecomposition according to any example described hereof promotes abalanced Th1/Th2 immune response to one or more allergens.

In another example, the composition according to any example describedhereof comprises an amount of killed H. pylori cells and/or inactivatedH. pylori cells and/or a cell lysate of said killed or inactivatedcells.

The present invention clearly extends to the manufacture of acomposition for use in preventing or treating allergy in a mammal, saidmanufacture comprising use of an isolated H. pylori cell, a cell lysatethereof, wherein said H. pylori cell is either killed or incapable ofcolonizing the mucosa of said mammal.

In one example, the present invention relates to use of an H. pyloricell such as an isolated H. pylori cell, and/or a cell lysate thereof ora combination thereof, wherein said H. pylori cell is inactivated orkilled in the preparation of a composition for preventing or treatingallergy in a mammal e.g., wherein the inactivated H. pylori cell doesnot have the same capacity of a live H. pylori cell having the samegenotype to colonize the mucosa of a mammal to which it is administeredor wherein the inactivated or killed H. pylori is incapable ofcolonizing the mucosa of a mammal to which it is administered.Optionally, wherein the cell lysate is a whole cell lysate (WCL) of theinactivated or killed H. pylori cell.

In another example, the present invention relates to use of aninactivated and/or killed H. pylori, such as an isolated and inactivatedand/or killed H pylori, or a cell lysate thereof or a combinationthereof in the preparation of a composition according to any exampledescribed hereof for interrupting or slowing or arresting or preventingan atopic march or progression of an atopic march in the subject.Optionally, wherein the cell lysate is a whole cell lysate (WCL) of theinactivated and/or H. pylori.

In another example, the present invention relates to use of aninactivated and/or killed H. pylori, such as an isolated and inactivatedand/or killed H. pylori, or a cell lysate thereof or a combinationthereof in the preparation of a composition according to any exampledescribed hereof for delaying or preventing or interrupting or slowingthe onset of one or more allergic conditions in the subject. Optionally,wherein the cell lysate is a whole cell lysate (WCL) of the inactivatedand/or killed H pylori.

In another example, the present invention relates to use of aninactivated and/or killed H. pylori, such as an isolated and inactivatedand/or killed H. pylori, or a cell lysate thereof or a combinationthereof in the preparation of a composition according to any exampledescribed hereof for delaying or preventing or interrupting or slowingthe onset of one or more of allergic eczema, urticaria, hives, rhinitis,wheezing, airway resistance, airway restriction, lung inflammation, foodallergy, or asthma. Optionally, wherein the cell lysate is a whole celllysate (WCL) of the inactivated and/or killed H. pylori cell.

In another example, the present invention relates to use of aninactivated and/or killed H. pylori, such as an isolated and inactivatedand/or killed H pylori, or a cell lysate thereof or a combinationthereof in the preparation of a composition according to any exampledescribed hereof for delaying or preventing or interrupting or slowingthe onset of airway resistance in response to an allergen. Optionally,wherein the cell lysate is a whole cell lysate (WCL) of the inactivatedand/or killed H. pylori.

In another example, the present invention relates to use of aninactivated and/or killed H. pylori, such as an isolated and inactivatedand/or killed H. pylori, or a cell lysate thereof or a combinationthereof in the preparation of a composition according to any exampledescribed hereof for delaying or preventing or interrupting or slowingthe onset of lung inflammation in response to an allergen. Optionally,wherein the cell lysate is a whole cell lysate (WCL) of the inactivatedand/or killed H. pylori.

In another example, the present invention relates to use of aninactivated and/or killed H. pylori, such as an isolated and inactivatedand/or killed H. pylori, or a cell lysate thereof or a combinationthereof in the preparation of a composition according to any exampledescribed hereof for delaying or preventing or interrupting or slowingthe cell infiltration into lung e.g., in response to an antigen.Optionally, wherein the cell lysate is a whole cell lysate (WCL) of theinactivated and/or killed H. pylori.

In another example, the present invention relates to use of aninactivated and/or killed H. pylori, such as an isolated and inactivatedand/or killed H. pylori, or a cell lysate thereof or a combinationthereof in the preparation of a composition according to any exampledescribed hereof for delaying or preventing or interrupting or slowingthe onset of an allergic condition characterized by an elevated serumlevel of allergen-specific IgE antibody and/or an elevated level of oneor more inflammatory cytokines in bronchioalveolar lavage (BAL) and/oran elevated level of cell infiltrate in lung. Optionally, wherein thecell lysate is a whole cell lysate (WCL) of the inactivated and/orkilled H. pylori.

Preferably, in the use according to any example described hereof, thecomposition is formulated for administration in absence of an adjuvantand does not include an adjuvant.

In one example, in the use according to any example described hereof,the composition is formulated for administration (e.g., by consumption)to infants, such as to infants who do not have developed lymphoidstructures and/or infants aged 0 to about 5 years. For example, whereinthe infants are aged between 0 to about 5 years, or between 0 to about 4years, or between 0 to about 3 years, or between 0 to about 2 years, orbetween 0 to about 1 year. In one example, the infants are aged between0 to about 2 years. In another example, the infants are aged betweenabout 4 months and about 12 months or between about 4 months and about18 months or about 4 months and about 24 months. In another example, theinfants are less than about 6 months of age.

In one example, in the use according to any example described hereofexample, the composition is formulated for administration (e.g., byconsumption) to a child older than about 5 years of age and/or toadolescents and/or to adults.

In one example, in the use according to any example described hereofexample, the composition is formulated for repeated administration, forexample, once per week, or twice per week, or three times per week, or 4times per week, or 5 times per week, or 6 times per week, or 7 times perweek, or more than 7 times per week, or more than twice per day. In onesuch example, the composition is formulated as a multi-dosage unitcomposition. For example, each dosage of the composition comprises anamount of the H. pylori bacteria or a lysate thereof in a rangecorresponding to between about 10² cells to about 10¹⁴ cells, or about10³ cells to about 10¹³ cells, or about 10⁴ cells to about 10¹³ cells,or about 10⁵ cells to about 10¹³ cells, or about 10⁶ cells to about 10¹³cells, or about 10⁶ cells to about 10¹² cells, or about 10⁷ cells toabout 10¹¹ cells, or about 10⁸ cells to about 10¹⁰ cells, or about 10⁹cells to about 10¹⁰ cells. For example, each dosage of the compositioncomprises an amount of the H. pylori bacteria or a lysate thereofcorresponding to about 10⁸ cells, or about 10⁹ cells, or about 10¹⁰cells.

In one such example, the composition is formulated for administrationdaily, wherein a daily dosage of said composition comprises an amount ofthe H. pylori bacteria or a lysate thereof in a range corresponding tobetween about 10² cells to about 10¹⁴ cells, or about 10³ cells to about10¹³ cells, or about 10⁴ cells to about 10¹³ cells, or about 10⁵ cellsto about 10¹³ cells, or about 10⁶ cells to about 10¹³ cells, or about10⁶ cells to about 10¹² cells, or about 10⁷ cells to about 10¹¹ cells,or about 10⁸ cells to about 10¹⁰ cells, or about 10⁹ cells to about 10¹⁰cells. For example, each daily dosage of the composition comprises anamount of the H. pylori bacteria or a lysate thereof corresponding toabout 10⁸ cells, or about 10⁹ cells, or about 10¹⁰ cells.

In one example, in the use according to any example described hereofexample, the dosage e.g., daily dosage of the composition is to beadministrated to a subject (e.g., by consumption) over a period of atleast about 2 weeks or at least about 4 weeks or at least about 6 weeksor at least about 8 weeks or at least about 10 weeks or at least about11 weeks or at least about 12 weeks or at least about 13 weeks at leastabout 14 weeks or at least about 15 weeks or at least about 16 weeks orat least about 17 weeks or at least about 18 weeks or at least about 19weeks or at least about 20 weeks or at least about 21 weeks or at leastabout 22 weeks or at least about 23 weeks or at least about 24 weeks orat least about 25 weeks, or at least about 6 months, or at least aboutone year or more than one year, preferably over a period of at leastabout 13 weeks or at least about 3 months.

In one example, in the use according to any example described hereof,the composition or a dosage (e.g., daily dosage) of the compositionpromotes a balanced development of an immune system in a juvenilesubject. In one example, in the use according to any example describedhereof, the composition or a dosage (e.g., daily dosage) of thecomposition promotes acquisition of adaptive immunity and/or innateimmunity in a subject. In one example, in the use according to anyexample described hereof, the composition or a dosage (e.g., dailydosage) of the composition promotes or enhances CD1d receptor activationand/or CD4-negative and CD8-negative natural killer (NK) cells in asubject. In one example, in the use according to any example describedhereof, the composition or a dosage (e.g., daily dosage) of thecomposition promotes or enhances γδ T-cell activation. In one example,in the use according to any example described hereof, the composition ora dosage (e.g., daily dosage) of the composition promotes or enhancesmucosal immunity involving immune recognition and presentation toantigen-presenting cells (APCs). In one example, in the use according toany example described hereof, the composition or a dosage (e.g., dailydosage) of the composition promotes a balanced Th1/Th2 immune responseto one or more allergens.

In one example, in the use according to any example described hereof,the composition comprises an amount of killed H. pylori cells and/orinactivated H. pylori cells and/or a cell lysate of said killed orinactivated cells.

The present invention also clearly extends to use of the compositionaccording to any example described hereof or to use of isolated H.pylori cell, a cell lysate thereof, wherein said H. pylori cell iseither killed or incapable of colonizing the mucosa of said mammal.

In one example, the present invention provides use of the composition asdescribed according to any example hereof in preventing or treatingallergy in a subject.

In another example, the present invention provides use of thecomposition according to any example described hereof in interrupting orslowing or arresting or preventing an atopic march or progression of anatopic march in a subject.

In another example, the present invention provides use of thecomposition according to any example described hereof in delaying orpreventing or interrupting or slowing the onset of one or more allergicconditions in a subject.

In another example, the present invention provides use of thecomposition according to any example described hereof in delaying orpreventing or interrupting or slowing the onset of one or more ofallergic eczema, urticaria, hives, rhinitis, wheezing, airwayresistance, airway restriction, lung inflammation, food allergy, orasthma.

In another example, the present invention provides use of thecomposition according to any example described hereof in delaying orpreventing or interrupting or slowing the onset of airway resistance inresponse to an allergen.

In another example, the present invention provides use of thecomposition according to any example described hereof in delaying orpreventing or interrupting or slowing the onset of lung inflammation inresponse to an allergen.

In another example, the present invention provides use of thecomposition according to any example described hereof in delaying orpreventing or interrupting or slowing cell infiltration into lung.

In another example, the present invention provides use of thecomposition according to any example described hereof in delaying orpreventing or interrupting or slowing the onset of an allergic conditioncharacterized by an elevated serum level of allergen-specific IgEantibody and/or an elevated level of one or more inflammatory cytokinesin bronchioalveolar lavage (BAL) and/or an elevated level of cellinfiltrate in lung.

In another example, the present invention provides use of atherapeutically effective amount of killed and/or inactivated H. pyloricells, or a cell lysate thereof or a combination thereof, in preventingor attenuating allergic airway hyper-responsiveness in lungs of asubject following exposure of the subject to an allergen. Preferably,wherein said use comprises use of a therapeutically effective amount ofkilled and/or inactivated H. pylori cells.

In another example, the present invention provides use of atherapeutically effective amount of killed and/or inactivated H. pyloricells, or a cell lysate thereof or a combination thereof, in preventingor attenuating allergic airway hyper-responsiveness in lungs of asubject following exposure of the subject to an allergen. Preferably,wherein said use comprises use of a therapeutically effective amount ofkilled and/or inactivated H. pylori cells.

In another example, the present invention provides use of atherapeutically effective amount of killed and/or inactivated H. pyloricells, or a cell lysate thereof or a combination thereof, in preventingor alleviating airway resistance in lungs of an asthmatic subjectfollowing exposure of said subject to an allergen. Preferably, whereinsaid use comprises use of a therapeutically effective amount of killedand/or inactivated H. pylori cells.

In another example, the present invention provides use of atherapeutically effective amount of killed and/or inactivated H. pyloricells, or a cell lysate thereof or a combination thereof, in preventingan allergic immune response to an allergen in a subject or reducingseverity or incidence of an allergic immune response to an allergen in asubject. Preferably, wherein said use comprises use of a therapeuticallyeffective amount of killed and/or inactivated H. pylori cells.

Preferably, in the use according to any example described hereof, the H.pylori or the lysate or the composition is used in absence of anadjuvant.

In yet another example, the present invention provides a method ofpreventing allergy in a mammal at risk of developing said allergycomprising the step of administering to said mammal an effective amountof a composition comprising an isolated H. pylori cell, a cell lysatethereof or combination thereof and a pharmaceutically accepted carrier,wherein said H. pylori cell is either killed or incapable of colonizingthe mucosa of said mammal, wherein said composition, uponadministration, provides protective immunity against said allergy.

Accordingly in one example, the present invention provides a method oftreating or preventing allergy in a mammalian subject, said methodcomprising administering the composition according to any exampledescribed hereof to a subject in need thereof.

In another example, the present invention provides a method ofpreventing or attenuating allergic airway hyper-responsiveness in lungsof a subject following exposure of the subject to an allergen, saidmethod comprising administering to the subject a therapeuticallyeffective amount of killed and/or inactivated H. pylori cells, or a celllysate thereof or a combination thereof, sufficient to prevent airwayhyper-responsiveness in a subject following exposure of said subject toan allergen. Preferably, wherein said method comprises administering atherapeutically effective amount of killed and/or inactivated H. pyloricells.

In another example, the present invention provides a method ofpreventing or alleviating airway resistance in an asthmatic subject,said method comprising administering to a subject in need thereof atherapeutically effective amount of killed and/or inactivated H. pyloricells, or a cell lysate thereof or a combination thereof, sufficient toprevent airway hyper-responsiveness in lungs of the subject followingexposure of said subject to an allergen. Preferably, wherein said methodcomprises administering a therapeutically effective amount of killedand/or inactivated H. pylori cells.

In another example, the present invention provides a method ofpreventing an allergic immune response to an allergen in a subject orreducing severity or incidence of an allergic immune response to anallergen in a subject, said method comprising administering to a subjectin need thereof a therapeutically effective amount of killed and/orinactivated H. pylori cells, or a cell lysate thereof or a combinationthereof. Preferably, wherein said method comprises administering of atherapeutically effective amount of killed and/or inactivated H. pyloricells.

In another example, the present invention provides a method ofinterrupting or slowing or arresting or preventing an atopic march orprogression of an atopic march in a subject, the method comprisingadministering the composition according to any example described hereofto the subject.

In another example, the present invention provides a method of delayingor preventing or interrupting or slowing the onset of one or moreallergic conditions in a subject, said method comprising administeringthe composition according to any example described hereof to thesubject.

In another example, the present invention provides a method of delayingor preventing or interrupting or slowing the onset of one or more ofallergic eczema, urticaria, hives, rhinitis, wheezing, airwayresistance, airway restriction, lung inflammation, food allergy, orasthma in a subject, the method comprising administering the compositionaccording to any example described hereof to the subject.

In another example, the present invention provides a method of delayingor preventing or interrupting or slowing the onset of airway resistancein response to an allergen in a subject, the method comprisingadministering the composition according to any example described hereofto the subject.

In another example, the present invention provides a method of delayingor preventing or interrupting or slowing the onset of lung inflammationin response to an allergen in a subject, the method comprisingadministering the composition according to any example described hereofto the subject.

In another example, the present invention provides a method of delayingor preventing or interrupting or slowing cell infiltration into lung ofa subject in response to an allergen, the method comprisingadministering the composition according to any example described hereofto the subject.

In another example, the present invention provides a method of delayingor preventing or interrupting or slowing the onset of an allergiccondition in a subject, wherein said condition is characterized by anelevated serum level of allergen-specific IgE antibody and/or anelevated level of one or more inflammatory cytokines in bronchioalveolarlavage (BAL) and/or an elevated level of cell infiltrate in lung of thesubject, and wherein the method comprising administering the compositionaccording to any example described hereof to the subject.

Preferably, in the method described according to any example hereof, theH. pylori or the lysate or the composition is administered in absence ofan adjuvant, and wherein the composition does not comprise an adjuvant.

In one example of the method according to any example described hereof,the composition or the inactivated or killed H. pylori cells and/or thelysate thereof is administered to the subject by the oral route (i.e.,for ingestion by the subject) and/or by inhumation.

In one example of the method according to any described hereof, thecomposition or the inactivated or killed H. pylori cells and/or thelysate thereof is administered (e.g., by consumption) to infants, suchas infants who do not have developed lymphoid structures and/or whereinthe infant is aged 0 to about 5 years. For example, the infants are agedbetween 0 to about 5 years, or between 0 to about 4 years, or between 0to about 3 years, or between 0 to about 2 years, or between 0 to about 1year. In one example, the infants are aged between 0 to about 2 years.In another example, the infants are in the age between about 4 monthsand about 12 months or between about 4 months and about 18 months orabout 4 months and about 24 months. In another example, the infants areless than about 6 months of age.

In another example, in the method according to any example describedhereof the composition or the inactivated or killed H. pylori cellsand/or the lysate thereof is administered (e.g., by consumption) to achildren older than about 5 years of age and/or to adolescents and/or toadults.

In another example, the method according to any example described hereofcomprises repeated administration of the composition or the inactivatedor killed H. pylori cells and/or the lysate thereof to the subject. Inone example, the composition or the inactivated or killed H. pyloricells and/or the lysate thereof is administered to the subject once perweek, or twice per week, or three times per week, or 4 times per week,or 5 times per week, or 6 times per week, or 7 times per week, or morethan 7 times per week, or more than twice per day.

In one such example, the method according to any example describedhereof comprises administering a dosage of the composition comprising anamount of the H. pylori bacteria or a lysate thereof in a rangecorresponding to between about 10² cells to about 10¹⁴ cells, or about10³ cells to about 10¹³ cells, or about 10⁴ cells to about 10¹³ cells,or about 10⁵ cells to about 10¹³ cells, or about 10⁶ cells to about 10¹³cells, or about 10⁶ cells to about 10¹² cells, or about 10⁷ cells toabout 10¹¹ cells, or about 10⁸ cells to about 10¹⁰ cells, or about 10⁹cells to about 10¹⁰ cells. For example, each dosage of the compositioncomprises an amount of the H. pylori bacteria or a lysate thereofcorresponding to about 10⁸ cells, or about 10⁹ cells, or about 10¹⁰cells. In one such example, the method according of the presentinvention according to any described hereof, comprises administering adaily dosage of the composition, wherein the wherein the daily dosage ofthe composition comprises an amount of the H. pylori bacteria or alysate thereof in a range corresponding to between about 10² cells toabout 10¹⁴ cells, or about 10³ cells to about 10¹³ cells, or about 10⁴cells to about 10¹³ cells, or about 10⁵ cells to about 10¹³ cells, orabout 10⁶ cells to about 10¹³ cells, or about 10⁶ cells to about 10¹²cells, or about 10⁷ cells to about 10¹¹ cells, or about 10⁸ cells toabout 10¹⁰ cells, or about 10⁹ cells to about 10¹⁰ cells. For example,each daily dosage of the composition comprises an amount of the H.pylori bacteria or a lysate thereof corresponding to about 10⁸ cells, orabout 10⁹ cells, or about 10¹⁰ cells.

In another example, the method according to any example described hereofcomprises administering a daily dosage of the H. pylori or the lysate orcomposition over a period of over a period of at least about 2 weeks orat least about 4 weeks or at least about 6 weeks or at least about 8weeks or at least about 10 weeks or at least about 11 weeks or at leastabout 12 weeks or at least about 13 weeks at least about 14 weeks or atleast about 15 weeks or at least about 16 weeks or at least about 17weeks or at least about 18 weeks or at least about 19 weeks or at leastabout 20 weeks or at least about 21 weeks or at least about 22 weeks orat least about 23 weeks or at least about 24 weeks or at least about 25weeks, or at least about 6 months, or at least about one year or morethan one year, preferably over a period of at least about 13 weeks or atleast about 3 months

In another example of the method according of the present inventionaccording to any example described hereof the administration or the H.pylori or the lysate or composition promotes development of a balanceddevelopment of an immune system in a juvenile subject.

In another example of the method according to any example describedhereof the administration or the H. pylori or the lysate or compositionpromotes acquisition of adaptive immunity in a subject.

In another example of the method according to any described hereof, theadministration or the H. pylori or the lysate or composition promotesacquisition of adaptive immunity in a subject.

In another example of the method according to any described hereof, theadministration or the H. pylori or the lysate or composition promotes orenhances CD1d receptor activation and/or CD4-negative and CD8-negativenatural killer (NK) cells.

In another example of the method according to any described hereof, theadministration or the H. pylori or the lysate or composition promotes orenhances γδ T-cell activation.

In another example of the method according of the present inventionaccording to any described hereof, the administration or the H. pylorior the lysate or composition promotes or enhances mucosal immunityinvolving immune recognition and presentation to antigen-presentingcells (APCs).

In another example of the method according to any example hereof, theadministration or the H. pylori or the lysate or composition promotes abalanced Th1/Th2 immune response to one or more allergens.

In another example of the method according to any described hereof, thesubject is asymptomatic for eczema, or asymptomatic for allergy, orasymptomatic for asthma, and wherein said method prevents a subsequentonset of eczema and/or allergy and/or asthma in the subject e.g.,following exposure of the subject to an allergen. In one such example,the method comprises administering an isolated and inactivated H. pylorito a juvenile subject to prevent eczema in the infant or a subsequentonset of allergy or asthma in later life. Alternatively, the methodcomprises administering the isolated and inactivated H. pylori to anadolescent or adult subject to prevent eczema in the subject or asubsequent onset of allergy or asthma in later life in the subject.However, a subsequent onset of eczema and/or allergy and/or asthma maybe induced in an untreated subject to whom the H. pylori or thecomposition has not been administered by exposure of the untreatedsubject to an allergen. For example, the allergen is an environmentalallergen, pollen allergen, dust mite allergen, animal allergen, orchemical allergen.

In another example of the method according to any described hereof, thesubject has suffered previously from one or more incidences of allergiceczema, allergy, or asthma, and wherein said method prevents asubsequent attack or reduces severity of a subsequent attack in thesubject. In one such example, the method comprises administering theinactivated and/or killed H. pylori, such as isolated inactivated and/orkilled H. pylori, or a cell lysate thereof or a combination thereof, toan adolescent or adult subject that has suffered previously fromallergic eczema and/or allergy and/or asthma, to thereby prevent asubsequent attack or reduce severity of a subsequent attack, optionallyto prevent or slow further atopic march in the subject. Alternatively,the method comprises administering the inactivated and/or killed H.pylori such as isolated inactivated and/or killed H. pylori, or a celllysate thereof or a combination thereof, to an adolescent or adultsubject that has suffered previously from allergic eczema and/or allergyand/or asthma, to thereby prevent a subsequent attack or reduce severityof a subsequent attack, optionally to prevent or slow further atopicmarch in the subject. However, a subsequent attack of eczema and/orallergy and/or asthma may be inducible in an untreated subject to whomthe H. pylori or the composition has not been administered by exposureof the untreated subject to an allergen. For example, the allergen is anenvironmental allergen, pollen allergen, dust mite allergen, animalallergen, or chemical allergen.

In another example of the method according to any described hereof,administration of an inactivated and/or killed H. pylori, such asisolated inactivated and/or killed H. pylori, or a cell lysate thereofor a combination thereof, to a subject reduces the incidence of allergicimmune responses in a population of subjects.

In another example of the method according to any described hereof,administration of an inactivated and/or killed H. pylori, such asisolated inactivated and/or killed H. pylori, or a cell lysate thereofor a combination thereof, to a subject reduces the incidence of allergicimmune responses in adolescent and/or adult members of the populationtreated when they were juveniles.

In some embodiments, the mammal is a naive mammal. Thus, in a furtherexample, the present invention provides a method of preventing allergyin an immunologically naive mammal at risk of developing said allergy,said method comprising the step of: (i) identifying a mammal at risk ofdeveloping an allergy; (ii) administering to said mammal a compositioncomprising an isolated H. pylori cell, a cell lysate thereof orcombination thereof and a pharmaceutically accepted carrier, whereinsaid H. pylori cell is either killed or incapable of colonizing themucosa of said mammal and (iii) allowing sufficient time to elapse toenable energy to develop.

In a further example, the present invention provides a method oftreating allergy in a mammal comprising the step of administering tosaid mammal an effective amount of a composition comprising an isolatedH. pylori cell, a cell lysate thereof or combination thereof and apharmaceutically accepted carrier, wherein said H. pylori cell is eitherkilled or incapable of colonizing the mucosa of said mammal, whereinsaid composition, upon administration, provides protective immunityagainst said allergy.

The mammal or subject includes a dog, a cat, a livestock animal, aprimate or a horse. In some embodiment, the mammal or subject is a humansubject. Preferably, the human subject is below the age of about 5. Morepreferably, the human subject is below the age of 2 years.

In one example, the present invention provides a kit for treating and/orpreventing allergy in a mammal comprising (i) a composition according toany example hereof and (ii) instructions for use in a method accordingto any one of examples hereof.

The present invention also clearly extends to a kit for treating and/orpreventing allergy in a subject, said kit comprising (i) the inactivatedand/or killed H. pylori or the lysate or the composition as describedaccording to any example hereof, and optionally (ii) instructions foruse in a method or use according to any one of examples hereof. Forexample, the kit is for use in preventing or attenuating allergic airwayhyper-responsiveness in lungs of a subject following exposure of thesubject, such as an asthmatic subject, to an allergen. Alternatively, orin addition, the kit is for use in preventing or alleviating airwayresistance or airway hyper-responsiveness in lungs of an asthmaticsubject following exposure of said subject to an allergen.Alternatively, or in addition, the kit is for use in preventing anallergic immune response to an allergen in a subject or reducingseverity or incidence of an allergic immune response to an allergen in asubject. Alternatively, or in addition, the kit is for use ininterrupting or slowing or arresting or preventing an atopic march orprogression of an atopic march in a subject. Alternatively, or inaddition, the kit is for use in delaying or preventing or interruptingor slowing the onset of one or more allergic conditions in a subject,for example wherein the one or more condition(s) is/are characterized byan elevated serum level of allergen-specific IgE antibody and/or anelevated level of one or more inflammatory cytokines in bronchioalveolarlavage (BAL) and/or an elevated level of cell infiltrate in lung of thesubject. Alternatively, or in addition, the kit is for use in delayingor preventing or interrupting or slowing the onset of one or more ofallergic eczema, urticaria, hives, rhinitis, wheezing, airwayresistance, airway restriction, lung inflammation, food allergy, orasthma in a subject. Alternatively, or in addition, the kit is fordelaying or preventing or interrupting or slowing the onset of airwayresistance and/or lung inflammation response to an allergen in asubject. Alternatively, or in addition, the kit is for delaying orpreventing or interrupting or slowing cell infiltration into lung of asubject in response to an allergen.

In a further example, the present invention provides a method ofgenerating a H. pylori strain that is able to provide protectiveimmunity against allergy comprising the steps of:

-   -   (a) providing an isolated H. pylori cell that is;        -   (i) incapable of colonizing the mucosa of a mammal and/or        -   (ii) cagA minus (cagA) and optionally positive for the            toxigenic s1 and m1 alleles of the vacA gene;    -   (b) optionally passaging said H. pylori cell through an animal        host; and    -   (c) optionally inactivating or killing said H. pylori cell.

Unless the context requires otherwise or specifically stated to thecontrary, integers, steps, or elements of the invention recited hereinas singular integers, steps or elements clearly encompass both singularand plural forms of the recited integers, steps or elements.

As used herein the term “derived from” shall be taken to indicate that aspecified integer may be obtained from a particular source albeit notnecessarily directly from that source.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated step or element orinteger or group of steps or elements or integers but not the exclusionof any other step or element or integer or group of elements orintegers.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter. Accordingly, as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. For example, a reference to “abacterium” includes a plurality of such bacteria, and a reference to “anallergen” is a reference to one or more allergens.

Each example described herein is to be applied mutatis mutandis to eachand every other example unless specifically stated otherwise.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specificexamples described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.However, publications mentioned herein are cited for the purpose ofdescribing and disclosing the protocols, reagents and vectors which arereported in the publications and which might be used in connection withthe invention. Nothing herein is to be construed as an admission thatthe invention is not entitled to antedate such disclosure by virtue ofprior invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, panel A, shows the “allergic (or atopic) march” which refers tothe typical progression of allergic diseases that often begin early inlife and illustrates the relative prevalence of clinical symptoms andmanifestations of allergic diseases according to age of individuals. Theallergic diseases include atopic dermatitis (eczema), food allergy,allergic rhinitis (hay fever) and asthma. In general, no clinicalsymptoms are detectable at birth. A majority of children with eczemawill progress to develop food allergies and/or allergic asthma, and asignificant proportion of these individuals will have atopic orrespiratory allergies as adults. Also, asthmatic wheezing may already beobserved during early infancy and although the majority of earlywheezers turn out to be transiently symptomatic, in some childrenwheezing may persist throughout school age and adolescence.

FIG. 1, panel B, shows the gradient or relative distribution of H.pylori in the gastrointestinal tract. H. pylori bacterial is a mammaliangut commensal organism that may be present in the gut alongside manyother bacteria. H pylori is generally acquired by the oral route andcolonized the gut, and may be asymptomatic in over 80% if humans,although persistent colonization of the stomach is associated withhigher risk of peptic ulcers, gastric cancers and other disorders suchas chronic urticarial (hives). H. pylori is continuously shed in largeamounts from the stomach into the lower intestines where it may be takenup by the Peyer's patches and may modulate the immune system via thePeyer's patches to establish persistent gastric infection (Gerirtz A Tand Sitaraman S V, 2007, Gastroenterology 133: 1044-1045; Nagi S et al.,2007, Proc Natl Acad Sci USA 109: 8971-8976; Watanabe N et al., 2008,Gastroenterology 134: 642-643). Active colonization by H. pylori maymodulate the host immune system towards immune tolerance of H. pylori toallow persistent colonization, and is associated with reduce risk ofallergic conditions in the host (Amedei A et al., 2010, J AsthmaAllergy. 3:139-147; Kosunen T U et al., 2002, Clin Exp Allergy.32:373-378; Chen and Blaster M J, 2007, Arch Intern Med. 167:821-827).

FIG. 1, panel C, shows an acute allergic asthma model using an OVAsensitization/challenge in which untreated H. pylori (marked “live Hp”)and treated H. pylori i.e., inactivated and/or killed H. pylori (marked“killed Hp”) are each administered to a mouse model of allergy andchallenged at the time points indicated.

FIG. 2 shows replication efficacy of live untreated H. pylori OND86control cells (marked “live”) and treated H. pylori i.e., inactivatedand/or killed H. pylori following treatment by UV-C irradiation (marked“UV”) and optionally heat treatment (marked “UV+HEAT”) or followingincubation for 48 hours under anaerobic conditions (marked “O₂Restriction”) and optionally heat treatment (marked “O₂Restriction+HEAT”). Replication efficacy of live and treated cells aredetermined by cells count i.e., colony forming units (CFU) measured onCBA plates after incubation of live and treated H. pylori i.e.,inactivated and/or killed H. pylori for 3 days at 37° C. in amicroaerobic environment containing 5% (v/v) CO₂ and less than 5% (v/v)O₂. Results obtained from two independent experiments are shown (marked“Repeat 1” and “Repeat 2”).

FIG. 3 shows percentage of urease activity in treated i.e., inactivatedand/or killed H pylori following heat treatment (marked “Heat”), UV-Cirradiation (marked “UV”) and optionally heat treatment (marked“UV+HEAT”) or following oxygen starvation (marked “O₂ Res”) andoptionally heat treatment (marked “O₂ Res+HEAT”) relative to ureaseactivity in untreated live H. pylori cells (marked “Live”).

FIG. 4 shows membrane redox potential ratio of live H pylori cells(marked “Live”) or H. pylori exposed to treatment by oxygen starvation(marked “O2r” or “O2R”) UV-C irradiation (marked “UV”) before and afterheat treatment (marked “heat”).

FIG. 5 shows that untreated H pylori (marked “live H. pylori”) andtreated H. pylori i.e., inactivated and/or killed H pylori (marked“killed H. pylori”) each improve outcomes of allergic asthma in the OVAmodel of allergic airways disease.

FIG. 6 shows that untreated H. pylori (marked “Hp”) and treated H.pylori i.e., inactivated and/or killed H. pylori (marked “killed”) eachreduce total cell counts (panel A) and eosinophilia (panel B) in the OVAmodel of allergic airways disease.

FIG. 7 shows a decreased OVA-specific IgE (panel A) and OVA-specific IgG(panel B) response in mice infected with either untreated H. pylori(marked “Hp”) and treated H. pylori i.e., inactivated and/or killed Hpylori (marked “killed”) in the allergic asthma model.

FIG. 8 shows that IL-13 is reduced in the lungs of mice infected witheither untreated H pylori (marked “live H pylori”) and treated H pylorii.e., inactivated and/or killed H. pylori (marked “killed”) in theallergic asthma model.

FIG. 9 shows the decreased number (panel A) and function (panel B) ofOVA-specific CD8 T cells in H. pylori infected mice (marked “live H.pylori”) compared to control mice (marked “naive”) after OVA/alumchallenge.

FIG. 10 shows decreased antigen-specific IgG in H. pylori-infected mice(marked “live H. pylori”) compared to control mice (marked “naive”),after primary (panel A) and secondary (panel B) OVA/alum challenge.

FIG. 11 shows the reduced responsiveness of CD4 (panel A) and CD8 T(panel B) cells from H. pylori infected mice (marked “live H. pylori”)compared to control mice (marked “naive”) in response to a non-specificstimulus.

FIG. 12 shows that H. pylori colonisation improves outcomes of allergicairways disease in the neonatal allergic asthma model. Panel A showsthat airway resistance increased in allergic adult mice not infectedwith live H. pylori, whereas mice challenged with live H. pylori fromday 5 exhibited comparable airway resistance to that of non-allergicmice. Panel B shows that H. pylori colonization prevents cellularinfiltration in the lungs after allergen challenge and that the totalcell count was similar to non-allergic control mice.

FIG. 13 shows that untreated H. pylori (marked “live H. pylori” in thex-axes) and treated H. pylori i.e., inactivated and/or killed H. pylori(marked “killed” in the x-axes) each improve immunological outcomes inthe neonatal allergic asthma model. Panel A shows that administration ofeither treated or live H. pylori effectively reduced cellularinfiltration in the lungs of H. pylori treated mice. Panel B shows thatadministration of either treated H. pylori or live H. pylori alsoreduced allergic allergen-specific IgE antibodies in H. pylori treatedsubjects. Panels C and D demonstrate that administration of treated (orlive) H. pylori was successful in reducing production of cytokinemediators and biological markers of asthma and allergic respiratorydisease, IL-5 and IL-13, in the lungs.

FIG. 14 shows that untreated H. pylori (marked “live Hp”) and treated H.pylori i.e., inactivated and/or killed H. pylori (marked “killed Hp”)are each effective in reducing allergic airway response to an allergenin adult and in neonatal mice. Panel A, shows results of airwayhyperresponsiveness (AHR) of lung tissue in response to increasing dosesof metacholine (MCh) challenge in adult mice infected with untreated H.pylori and treated H. pylori i.e., inactivated and/or killed H. pylori.Allergic adult mice controls which did not receive H. pylori i.e., wereuninfected, sensitised and challenged were marked “Positive” anduntreated healthy adult mice controls which did not receive H. pylorii.e., were uninfected and were only sensitised were marked “Negative”.Panel B, shows results of airway hyperresponsiveness (AHR) of lungtissue in response to increasing doses of metacholine (MCh) challenge inneonatal mice infected with untreated H. pylori. Panel C, shows resultsof airway hyperresponsiveness (AHR) of lung tissue in response toincreasing doses of metacholine (MCh) challenge in neonatal miceinfected with untreated H. pylori and with treated H. pylori i.e.,inactivated and/or killed H. pylori. In panels A and B, allergic adultneonatal mice controls which did not receive H. pylori i.e., wereuninfected, sensitised and challenged were marked “Positive”, anduntreated healthy neonatal mice controls which did not receive H. pylorii.e., were uninfected and were only sensitised were marked “Negative”.The results shown in panels A, B and C represent three independentexperiments.

FIG. 15 shows results of colonization efficacy of untreated H. pylori(marked “live”) and treated H. pylori i.e., inactivated and/or killed H.pylori (marked “treated”) in allergic subjects in the adult allergicasthma model.

FIG. 16 shows results of colonization efficacy in mice of inactivatedand/or killed H. pylori produced by treatment of live OND79 H. pyloricells with UV-C irradiation (marked “OND79 UV”) and optionally heattreatment (marked “OND79 UV+HEAT”) or by oxygen starvation (marked“OND79 O2R”) and optionally heat treatment (marked “OND79 O2R+HEAT”).Colonization efficacy is shown as the number of colony forming unity(CFU) detected in stomach of infected mice.

FIG. 17 shows that UV treated i.e., inactivated and/or killed H. pyloriOND79 (marked “OND79”) and H. pylori J99 (marked “J99”) strains ofdifferent origins were effective in reducing allergen (OVA)-specific IgE(Panel A) and IgG (Panel B) antibodies in neonatal allergic asthma mousemodel. Control mice were uninfected, sensitised and challenged (positivecontrol i.e., untreated allergic mice; marked “Pos”) or only sensitised(negative control i.e., untreated healthy mice; marked “Neg”). Titres ofOVA-specific antibodies were measured in mice serum diluted 1:60, andexpressed as the individual and average absorbance at OD405 nm.

FIG. 18 shows randomly amplified polymorphic DNA (RAPD) analysis of asingle colony isolate of H. pylori OND79 and single colony isolates sixclinical isolates of a derivative H. pylori obtained from a gastricbiopsy sample following passaging of H. pylori OND79 in a human host.The six clinical isolates of the derivative H. pylori were labelled“#1157 clone 1”, “#1157 clone 9”, “#86198 clone 1”, “#86198 clone 9”,“#45156 clone 1” and “#45156 clone 9”. Genetic fingerprinting wasperformed as described by Akopyanz et al., (1992) Nucleic AcidsResearch, 20:5137-5142 using the primer “1254” set forth in SEQ ID NO: 3and having the sequence 5′-CCG CAG CCA A-3′ (Panel A), or the primer“1281” set forth in SEQ ID NO: 4 and having the sequence 5′-AAC GCG CAAC-3′ (Panel B). In each of Panel A or Panel B: lane M, 1 kilo base (kb)DNA ladder marker (New England Biolabs Inc., Ipswich, Mass., US); lane1, OND79 (parent strain); lane 2, #1157 clone 1; lane 3, #1157 clone 9;lane 4, #86198 clone 1; lane 5, #86198 clone 9; lane 6, #45156 clone 1;lane 7, #45156 clone 9. Genetic fingerprinting was identical for theparent input strain OND 79 and for each clinical isolate of thehuman-passaged derivative strain.

FIG. 19 shows results of infection and colonization efficacy in mice ofsix clinical isolates of H. pylori obtained after passaging H. pyloriOND79 in a human host. Infection and colonization efficacy of theclinical isolates is shown as the number of colony forming unity (CFU)detected in stomach of infected mice. The six H. pylori clinicalisolates are labelled “#1157 clone 1”, “#1157 clone 9”, “#86198 clone1”, “#86198 clone 9”, “#45156 clone 1” and “#45156 clone 9”. The H.pylori clinical isolate #1157 clone 9 corresponds to H. pylori OND86strain deposited under NMI Accession No. V14/013016.

FIG. 20 shows efficacy of six clinical isolates of H. pylori obtainedafter passaging H. pylori OND79 in a human host, to induce specificanti-H. pylori IgG antibody response 2 weeks after oral administrationof the isolates in the C57BL/6 mouse model. Antibody response titres areexpressed as the OD value measured at 405 nm. The six H. pylori clinicalisolates are labelled “#1157 clone 1”, “#1157 clone 9”, “#86198 clone1”, “#86198 clone 9”, “#45156 clone 1” and “#45156 clone 9”. The H.pylori clinical isolate #1157 clone 9 corresponds to H. pylori OND86strain deposited under NMI Accession No. V14/013016.

FIG. 21 shows safety and tolerability study in allergic adult subjectsincluding a dose escalation assessment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

1. H. pylori

The present invention provides for compositions comprising inactivatedand/or killed H. pylori or a cell lysate thereof.

In one example, the H. pylori is an inactivated H. pylori. The term“inactivated H. pylori” shall be taken to mean a cell or strain of H.pylori which does not have the same capacity of a live H. pyloribacterium having the same genotype to induce gastric ulcer or otherpathology such as a malignancy and/or does not have the samecolonization capability of a live bacterium H. pylori bacterium havingthe same genotype and/or does not have a functional or intact genome ofa live H. pylori bacterium having the same genotype. For example, aninactivated H. pylori may not have an intact genome yet retain afunctional transcriptome and/or translational machinery such that itretains at least portion of the metabolic activity of the correspondinglive H. pylori. Thus, it is preferred that an inactivated H. pyloriretains partial or full metabolic activity of a corresponding live H.pylori. By “live H. pylori” in this context is meant H. pylori that hasnot been treated as described according to any example hereof so as torender it inactive and/or killed.

Notwithstanding that an inactivated H. pylori may transiently associatewith the gastric mucosa of a mammal, it is preferred that suchinactivated bacteria are incapable of colonizing the gastric mucosa of amammal so as to establish chronic or persistent infection of the gastricmucosa. For example, inactivated H. pylori may have an impaired abilityto induce one or more H. pylori-associated pathogenic effects including,but not limited to, formation of peptic ulcers, gastric cancers such asnon-cardiac gastric adenocarcinoma or MALT lymphoma, and other disorderssuch as chronic urticarial (hives) that is normally associated withpersistent H. pylori colonization of the mucosa.

Preferably, an inactivated H. pylori retains the cell structure of liveH. pylori. For example, an inactivated H. pylori retains the structuralintegrity of the bacterial cell wall and/or cell membrane of live H.pylori such that it may not be disrupted or lysed.

Alternatively, or in addition, the inactivated H. pylori retains anability of a live H. pylori to be taken up by the Peyer's patches in thelower intestine of a mammal. For example, the inactivated H. pylori mayretain the immunogenicity and/or antigenicity and/or receptor-ligandinteraction of a corresponding live H. pylori having replicative andcolonizing functionalities.

Alternatively, or in addition, an inactivated H. pylori undergoes one ormore metabolic changes e.g., enhanced lipopolysaccharide synthesis andsurface presentation thereof and/or enhanced degradation of cellularproteins and/or reduced urease production during and/or following theirinactivation.

In another example, the H. pylori is a killed H. pylori. The term“killed H pylori” shall be taken to mean a cell or strain of H. pylori,which is irreversibly metabolically inactive. For example, a killed H.pylori is incapable of inducing gastric ulcer or other pathology such asa malignancy and/or is incapable of colonizing the gastric mucosa of amammal and/or does not have a functional or intact genome of a live H.pylori bacterium having the same genotype. Thus it is preferred that akilled H. pylori does not retain a functional transcriptome and/ortranslational machinery.

Preferably, a killed H. pylori retains the cell structure of an a liveH. pylori. For example, a killed H. pylori retains the structuralintegrity of the bacterial cell wall and/or cell membrane of aninactivated or live H. pylori such that it may not be disrupted orlysed.

Alternatively, or in addition, a killed H. pylori retains the ability ofa live H. pylori to be taken up by the Peyer's patches in the lowerintestine of a mammal. For example, the killed H. pylori may retain theimmunogenicity and/or antigenicity and/or receptor-ligand interaction ofa corresponding live H. pylori having replicative and colonizingfunctionalities.

In another example, the present invention employs H. pylori that hasbeen subjected to a process for inactivating the bacterium and a processfor killing H. pylori. For example, killing of cells captures thebenefits of the inactivated cells to the immune system following theiradministration whilst ensuring added safety of the organism for humanuse.

In the present context, a “cell lysate” is a preparation made frominactive and/or killed H. pylori cells as described according to anyexample hereof in which the inactive and/or killed H. pylori cells havebeen disrupted such that the cellular components of the bacteria aredisaggregated or liberated from the bacterial cell.

Persons skilled in the art are aware of means for producing bacterialcell lysates. For example, H. pylori cells are pelleted and thenresuspended in, for example, Dulbecco's phosphate buffered saline (PBS;10 mM phosphate, 0.14 M NaCl, pH 7.4) and subjected to sonication on icewith a W-375 sonication Ultrasonic processor (Heat Systems-Ultrasonics,Inc., Farmingdale, N.Y.) at 50% duty cycle with pulse and strengthsetting 5 for three 1 min sessions. If required, insoluble material andunbroken bacterial cells can then be removed by centrifugation.Alternatively, H pylori cells are pelleted and then resuspended in alysis buffer containing 25 mM Tris, pH 7.5, 1 mM MgCl₂, 1 mMaminopolycarboxylic acid (EGTA), 150 mM NaCl, 1% v/v nonylphenoxypolyethoxyl ethanol (e.g., Tergitol-type NP-40 from Sigma-AldrichInc.,) and 1% v/v protease and/or phosphatase inhibitor(s). The wholecell lysate is collected e.g., using a cell scraper and centrifuged at1,200 g, 4° C. for 15 min. Alternatively, H. pylori cells are collectedby centrifugation and resuspended in PBS and then lysed by passagethrough a French press (SLM Instrument Inc., Urbana, Ill.) at 20,000LB/in. Again, if required, the bacterial lysate are centrifuged at102,000×g for 10 minutes to remove bacterial debris and/or filteredthrough a 0.45 μM membrane (Nalgene, Rochester, N.Y.). Another method ofproducing cell lysate of H. pylori involves one or more cycles offreezing and thawing of bacterial pellets e.g., in the presence oflysozyme. A particular example of a H. pylori cell lysate is the solublefraction of a sonicated culture of the inactivated H. pylori, e.g.,obtained after filtration. Alternatively or in addition, H. pylori cellsare fragmented using a high-pressure homogenizer (e.g., Avestin modelEmulsiFlexC5). Optionally, the cell lysate is further treated usingformalin. In one example, the whole cell lysate (WCL) of H. pylori e.g.,obtained as described herein, is subjected to additional fractionationand/or purification to isolate or purify or separate one or morecomponents from the H. pylori cell lysate, such as cell proteins and/orlipids.

In another example, the live and/or inactivated and/or killed H. pylorimay be in an isolated form. As used herein the term “isolated” when usedin reference to H. pylori such as live and/or inactivated and/or killedH. pylori refers to H. pylori or cell or strain thereof present in anenvironment which is different to the native environment in which a liveH. pylori is naturally present. For example, the isolated H. pylori maybe removed or isolated from its native environment and/or substantiallyfree of at least one component found in the native environment of a liveH. pylori. The term “isolated” in this context includes a H. pylori cellculture, a partially-pure H. pylori cell preparation, and asubstantially pure H. pylori cell preparation.

In one particular example, H. pylori is provided in biologically-pureform. As used herein the term “biologically-pure” refers to an in vitroor ex vivo culture of H. pylori that is substantially free from otherspecies of microorganisms. Optionally, a biologically-pure H. pylori isin form of a culture of a single strain of H. pylori.

In yet another example, a killed and/or inactivated H pylon may comprisea cell lysate. For example, the cell lysate may be a whole cell lysateof H. pylori.

Alternatively, the present invention is employed using a compositioncomprising a mixture of the inactivated and/or killed H. pylori asdescribed according to any example hereof and a cell lysate, such as awhole cell lysate of an inactivated and/or killed H. pylori as describedaccording to any example hereof.

2. Cultivating H. pylori

Strains

Any H. pylori strain known in the art may be used in the preparation ofthe H. pylori compositions of the present invention. In one example, theH. pylori strain may be any live H. pylori strain deposited with anInternational Depositary Authority under the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure. For example, the H. pylori strain may beobtained from the American Type Culture Collection (ATCC) such as, forexample, H. pylori deposited under Accession No. ATCC 43504 or AccessionNo. ATCC 26695 or Accession No. ATCC BAA-945 or Accession No. ATCC700392 or Accession No. ATCC 49503 or Accession No. ATCC 53726 orAccession No. 53727 or Accession No. ATCC 43526 or Accession No. ATCC:43579 or accession number ATCC 700824. For example, H. pylori strain maybe J99 stain (ATCC 700824). Alternatively, or in addition, exemplary H.pylori strains are as described by Moodley Y et al., (2009), Science,323: 527-530 and/or by Falush D, et al., (2003), Science, 299:1582-1585. Alternatively, or in addition, exemplary H. pylori strainsthat may be used in the preparation of the compositions of the presentinvention were deposited with the National Measurement Institute (NMI),1/153 Bertrie Street, Port Melbourne, Victoria, Australia, pursuant tothe provisions of the Budapest Treaty as follows:

H. pylori strain name NMI Accession No Date of deposit OND737 V09/00910122 Apr. 2009 OND738 V09/009102 22 Apr. 2009 OND739 V09/009103 22 Apr.2009 OND740 V09/009104 22 Apr. 2009 OND248 V10/014059 28 May 2010 OND256V10/014060 28 May 2010 OND79 V13/023374 28 Nov. 2013 OND86 V14/013016 10Jun. 2014

In another example, the H. pylori may be any H. pylori clinical isolateobtained from mammalian e.g., human gastric biopsy samples from patientsdiagnosed to be infected with H. pylori such as those exhibiting chronicgastritis, peptic ulcers e.g., gastric and duodenal ulcers, and/orgastric malignancies. In one such example, the H. pylori bacteria in thepatient biopsy is inoculated onto a suitable culture medium such asColumbia agar containing 5% sheep blood (Invitrogen) and grown at 37° C.in a microaerophilic chamber (Don Whitley, West Yorkshire, UK) in 10%CO₂, 5% O₂, and 85% N₂; for example as described by Cheng-Chou Yu etal., (2013), PLoS ONE, 1: e55724. In another example, the H. pyloribacteria in the patient biopsy is inoculated onto H. pylori selectivemedia such as F12 agar medium plates comprising DENT's supplement,nalidixic acid and bacitracin e.g., commercially available fromThermoscientific, Australia. In one such example the H. pylori strain isTA1 (Cag+ and VacA+) as described by Cheng-Chou Yu et al., (2013) supra.

Accordingly, in some examples, the H. pylori strain of the presentinvention has been passaged through an animal host such as a human. Forexample, the H. pylori strain of the present invention is derived fromthe H. pylori strain OND79 after passage of the OND79 strain in a humansubject e.g., following infection and/or colonization of the gastricmucosa of a human subject with H. pylori OND79 strain. In one suchexample the H. pylori strain is obtained from a human gastric biopsysample of a human subject who has been administered with OND79 cells.For example, the H. pylori strain of the present invention is OND86.

Culture Media

Media used for cultivating H. pylori for bacterial growth and/ormaintenance are prepared by procedures known to the skilled artisan anddescribed, for example, in BD Diagnostics (Manual of MicrobiologicalCulture Media, Sparks, Md., Second Edition, 2009); Versalovic et al. (InManual of Clinical Microbiology, American Society for Microbiology,Washington D.C., 10th Edition, 2011), Garrity et al. (In Bergey's Manualof Systematic Bacteriology, Springer, New York, Second Edition., 2001);Ndip et al. 2003 J. Pediatr. Gastroenterol. Nutr. 36: 616-622 andTesterman et al. 2001 J. Clin. Microbiol. 39: 3842-3850. As will beapparent to the skilled artisan, H. pylori morphology may be assessed byperforming gram staining (See e.g. Coico 2005 Curr. Protoc. Micorbiol.Appendix 3) and viability of may be assessed using colony counts asdescribed, for example, by Murray et al. (In: Manual of ClinicalMicrobiology, American Society for Microbiology, Washington D.C., NinthEdition 2007).

A preferred cell culture medium is supplemented with bovine serum, or amodified cell culture medium comprising a serum alternative suitable forcultivation of mammalian cells and comprising sufficient carbon andenergy sources to support growth of H. pylori in a fermentation process.Preferred cell culture media have regulatory approval for use inproduction of human therapeutics.

For example, H. pylori may be cultured on a defined medium supplementedwith bovine serum and fortified with Fe3+. Exemplary medium is an F12liquid medium supplemented with NaHCO₃, fetal bovine serum (FBS) 10%(v/v) and FeSO₄ (75 μM).

In a particularly preferred example, medium for cultivation of H pyloricomprises calcium chloride (e.g., calcium chloride anhydrous), cupricsulfate (e.g., as cupric sulfate.5H₂O), ferrous sulfate (e.g.,FeSO₄.7H₂O), magnesium chloride (e.g., magnesium chloride anhydrous),potassium chloride, sodium chloride, sodium phosphate (e.g., sodiumphosphate dibasic [anhydrous]), zinc sulfate.7H₂O (e.g., zincsulfate.7H₂O), L-alanine, L-arginine (e.g., L-arginine.HCl),L-asparagine (e.g., L-asparagine. H₂O), L-aspartic acid, L-cysteine(e.g., L-cysteine.HCl or L-cysteine. HCl.H₂O), L-glutamic acid,L-glutamine, glycine, L-histidine (e.g., L-histidine. HCl orL-histidine.HCl.H₂O), L-isoleucine, L-leucine, L-lysine (e.g., L-lysine.HCl), L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine (e.g., L-tyrosine 2Na.2H₂O), L-valine,D-biotin, choline chloride, folic acid, myo-inositol, niacinamide,D-pantothenic acid (hemicalcium), pyridoxine (e.g., pyridoxine.HCl),riboflavin, thiamine (e.g., thiamine.HCl), vitamin B-12, D-glucose,hypoxanthine, linoleic acid, phenol red (e.g., phenol red.Na),putrescine dihydrochloride, pyruvic acid (e.g., pyruvic acid.Na)thioctic acid, thymidine and bovine serum.

In one such preferred example, the medium for cultivation of H. pyloricomprises 0.0333 g/L calcium chloride (anhydrous), 0.0000025 g/L cupricsulfate.5H₂O, 0.000834 g/L ferrous sulfate.7 H₂O, 0.0576 g/L magnesiumchloride [anhydrous], 0.224 g/L potassium chloride, 7.599 g/L sodiumchloride, 0.14204 g/L sodium phosphate dibasic (anhydrous), 0.000863 g/Lzinc sulfate.7H₂O, 0.009 g/L L-alanine, 0.211 g/L L-arginine. HCl,0.01501 g/L L-asparagine.H₂O, 0.0133 g/L L-aspartic acid, 0.035 g/LL-cysteine.HCl.H₂O, 0.0147 g/L L-glutamic acid, 0146 g/L L-glutamine,0.00751 g/L glycine, 0.02096 g/L L-histidine.HCl.H₂O, 0.00394 g/LL-isoleucine, 0.0131 g/L L-leucine, 0.0365 g/L L-lysine.HCl, 0.00448 g/LL-methionine, 0.00496 g/L L-phenylalanine, 0.0345 g/L L-proline, 0.0105g/L L-serine, 0.0119 g/L L-threonine, 0.00204 g/L L-tryptophan, 0.00778g/L L-tyrosine 2Na.2H₂O, 0.0117 g/L L-valine, 0.0000073 g/L D-biotin,0.01396 g/L choline chloride, 0.00132 g/L folic acid, 0.018 g/Lmyo-inositol, 0.000037 g/L niacinamide, 0.00048 g/L D-pantothenic acid(hemicalcium), 0.000062 g/L pyridoxine.HCl, 0.000038 g/L riboflavin,0.00034 g/L thiamine.HCl, 0.00136 g/L vitamin B-12, 1.802 g/L D-glucose,0.00408 hypoxanthine, 0.000084 g/L linoleic acid, 0.0013 g/L phenolred.Na, 0.000161 g/L putrescine dihydrochloride, 0.11 g/L pyruvicacid.Na, 0.00021 g/L thioctic acid, 0.00073 g/L thymidine and bovineserum 100 ml.

In media for culturing H. pylori, FBS may be substituted for bovineserum albumin with or without lipid supplementation. Alternatively,plasma may be substituted for FBS, because plasma comprises componentsof the coagulation cascade that may influence the physiology of thecells e.g., their lipid profile and/or protein profile and/or LPSprofile.

Other semi-synthetic media, based on plant proteins or other cellculture media, may also be employed.

H. pylori may be cultivated in a liquid, semisolid or solid form.Examples of liquid media include Brucella Broth, Columbia Broth, brainheart infusion broth, Wilkins-Chalgren broth, Ham's F-10 nutrient media,Ham's F-12 nutrient media, Mueller-Hinton broth, Skirrow Campylobactermedia, Belo Horizonte media, Dent's CP media and H. pylori specialpeptone broth as described for example by Stevenson et al. 2000 Lett.Appl. Microbiol. 30: 192-196. Semisolid and solid media may be preparedfrom any of the liquid media described in any example hereof, by theaddition of a solidifying agent such as, for example, agar.Alternatively, a specialised semisolid and/or solid medium may be used,such as, for example, Chocolate agar, Tryptic Soy Agar, Glupszynski'sBrussels campylobacter charcoal agar and Johnson-Murano agar.

The medium may be supplemented with blood or a blood component. As usedherein term “blood” shall be taken to mean whole blood and “bloodcomponent” refers to serum and/or plasma and/or plasma fractions and/orred blood cells and/or white blood cells and/or platelets and/or proteinfractions. Preferably, the blood or blood component is defibrinated. Inone example, the blood component is from a mammal.

Suitable mammals include, for example, goats, sheep, bison, cows, pigs,rabbits, buffalos, horses, rats, mouses, or humans. In a preferredexample, the blood component may comprise serum. In one example, theserum may be fetal calf serum or newborn calf serum or bovine serum.Media may be supplement with blood or a blood product at finalconcentration in the media of 1% (vol/vol) or at least 2% (vol/vol) orat least 3% (vol/vol) or at least 4% (vol/vol) or at least 5% (vol/vol)or at least 6% (vol/vol) or at least 7% (vol/vol) or at least 8%(vol/vol) or at least 9% (vol/vol) or at least 10% (vol/vol) or at least15% (vol/vol) or at least 20% (vol/vol) or at least 25% (vol/vol). Inanother example, the blood may comprise heat inactivated blood. Inanother example, the medium may comprise a mixture of heat inactivatedand non-heat inactivated blood. Methods of heat inactivating blood areknown in the art and are described, for example in Ayache et al. 2006 J.Transl. Med. 4:40.

Alternatively, H. pylori may be cultivated in blood-free medium, such asan egg yolk emulsion medium as described, for example, by Westblom etal. 1991 J. Clin. Microbiol. 29:819-821, or a cyanobacterial extractbased medium as described for example, by Vega et al. 2003 J. Clin.Micrbiol. 41: 5384-5388.

In another example, the medium may be supplemented with chemicalsupplements, such as for example, adenine and/or cysteine hydrochlorideand/or cyclodextrin and/or ferric nitrate and/or ferrous sulfate and/orpeptone and/or IsoVitaleX and/or Vitox and/or starch and/or sodiumbicarbonate and/or sodium pyruvate and/or mucin and/or Vitamin B12and/or L-glutamine and/or guanine and/or p-aminobenzoic acid and/orL-cystine and/or yeast extract.

In yet another example, the medium may be supplemented with antibioticscapable of inhibiting growth of non-H. pylori microorganisms. Suitableantibiotics may include, vancomycin and/or trimethoprim and/orcefsulodin and/or amphotericin B and/or polymyxine.

Preferably, H. pylori is cultured in medium without antibiotics.

Environmental Conditions

As will be known to the skilled artisan, H. pylori may be cultivated ina micro-aerobic atmosphere such as, for example, in a CO₂ incubator orin an anaerobic chamber with a micro-aerobic atmosphere or in a gas jarwith gas-generation kits as described.

Suitable micro-aerobic atmospheres are described, for example, by Mobleyet al. (In H. pylori: Physiology and Genetics. American Society forMicrobiology, Washington D.C., 2001). In one example, H. pylori may becultivated in an atmosphere comprising about 1% to about 10% oxygen,about 5% to about 10% carbon dioxide, and about 0% to about 10%hydrogen.

Temperature conditions used to cultivate H. pylori are known in the art.For example, H. pylori may be cultivated at a temperature of betweenabout 25° C. to about 45° C. Preferably, H. pylori may be cultivated ata temperature of between about 30° C. to about 40° C. More preferably,H. pylori may be cultivated at a temperature of about 37° C.

In one example, H. pylori may be stressed during cultivation. As usedherein, the term “stressed” shall be taken to mean a change in anenvironmental condition. For example, H. pylori may be exposed toenvironmental stresses such as, for example, oxidative stress, pHstress, osmotic stress, carbon starvation, phosphate starvation,nitrogen starvation, amino acid starvation, oxygen stress e.g., bygrowing H. pylori under anaerobic conditions, heat or cold shock ormutagenic stress. Preferably, exposure of H. pylori to environmentalstress(es) during cultivation results in one or more metabolic changesin H. pylori such as enhanced lipopolysaccharide synthesis and surfacepresentation thereof and/or degradation of H. pylori cellular proteins.

Cell Culture Containers

H. pylori may be cultivated in using standard cell culture containersknown to the skilled artisan, such as, for example multi-well plates,petri-dish, roller bottles, T flasks, D flasks, culture chambers,hyperflask vessels, spinner flasks and Erlenmeyer flasks.

Preferred cell culture conditions are optimized for cell culture medium,shear sensitivity, oxygen and other gas requirements, and pH control, toprovide for optimum growth of H. pylori in large-scale culture e.g., ahigh optical density of cell culture in a short time frame.

Preferably, H. pylori may be cultivated in a bioreactor. As used hereinthe term “bioreactor” shall be taken to mean an apparatus for thecultivation of prokaryotic and/or eukaryotic cell cultures undercontrolled conditions. The bioreactor may be operated in a batch or fedbatch or an extended batch or a repetitive batch or a draw/fill or arotating-wall or a spinning flask or a semi-continuous or perfusion or acontinuous mode.

In one example, the bioreactor may agitate the cell culture for purposesof aeration using methods such as, for example, rocking, stirring, orchanneling fluid or gas through the culture. Examples of suchbioreactors include, for example, stirred tank fermentors or bioreactorsagitated by rotating mixing devices, chemostats, bioreactors agitated byshaking devices, airlift fermentors/bioreactors, fluidized bedbioreactors, bioreactors employing wave induced agitation, centrifugalbioreactors or roller bottles.

In another example, the bioreactor may comprise means for quantificationof biomass, such as, for example, by measuring the optical density ofthe culture medium. Suitable means for quantification of biomassinclude, for example, an optical sensor or a waveguide sensor or a Ramanspectroscopy.

In yet another example, the bioreactor may include means for monitoringand/or measuring and/or adjusting one or more bioprocess parameters. Asused herein, the term “bioprocess parameter” shall be taken to mean achemical or physical property that may alter the growth rate of H.pylori. Suitable bioprocess parameters include, for example,temperature, pH, dissolved oxygen, carbon dioxide concentration, carbonsource concentration, bile salt concentration, light, glucoseconcentration, pressure, concentration of an ionic species,concentration of a cellular metabolite, molarity, osmolality, glucoseconcentration, serum concentration and degree of agitation.

As will be apparent to the skilled artisan, a number of methods may beused to determine the growth rate of H. pylori.

Preferably, the bioreactor is a microreactor. The term “microreactor” asused herein refers to a bioreactor having a volume of less than 1000 mLor less than 900 mL or less than 800 mL or less than 700 mL or less than600 mL or less than 500 mL or less than 400 mL or less than 300 mL orless than 200 mL or less than 100 mL or less than 90 mL or less 80 mL orless than 70 mL or less than 60 mL or less than 50 mL or less than 40 mLor less than 30 mL or less than 20 mL or less than 15 mL or less than 10mL or less than 9 mL or less than 8 mL or less than 7 mL or less than 6mL or less than 5 mL or less than 4 mL or less than 3 mL or less than 2mL or less than 1 mL. Commercially available microreactors include, forexample, the micro-Matrix (Applikon Biotechnology), the micro-flask(Applikon Biotechnology) and the advanced micro-scale bioreactor (TapBiosystems).

Alternatively, the bioreactor is a large scale bioreactor. As usedherein the term “large scale bioreactor” refers to a bioreactor used toproduce a product for sale or for production of an intermediate of aproduct for sale. Preferably, a large scale bioreactor has an internalcapacity of at least 1 L at least 2 L at least 3 L at least 4 L at least5 L at least 6 L at least 7 L at least 8 L at least 9 L at least 10 L atleast 20 L at least 50 L at least 100 L at least 200 L at least 300 L atleast 400 L at least 500 L at least 600 L at least 700 L at least 800 Lat least 900 L at least 1000 L in particular at least 2000 L at least3000 L or at least 4000 L.

In a particularly preferred example, H. pylori is cultured from a frozenor unfrozen glycerol stock or other liquid stock or plate stock,employing H. pylori e.g., in a stock volume of about 3 mL that is thenseeded into and cultured in a multichannel miniature bioreactor systemor scalable stirred tank bioreactor e.g., a 2 L bench-top stirred tankbioreactor. A seed train may be employed, wherein an inoculum isprepared for a pilot-scale bioreactor. For example, a 400 L pilot-scalebatch of H. pylori may be produced from one or two or three or four orfive seed stages wherein each seed stage provides a 10-foldamplification of bacterial culture density as determined by OD at about600 nm. In a similar scale-up process, an inoculum from a pilot-scalebioreactor is employed to inoculate a production-scale bioreactor. Forexample, batches of 2 L to 20 L of culture from a pilot-scale bioreactorprocess are combined until an appropriate volume is obtained forinoculation of a production-scale bioreactor. Incubation times for eachstage vary in a range from about 16 hours to about 120 hours, including16 hours to about 96 hours.

In another example, a seed culture is used to amplify cells and processvolume to generate an inoculum for a pilot-scale bioreactor, which isthen employed to inoculate medium in a production-scale bioreactor. Forexample, H. pylori cells (0.5 mL) stored frozen at −80° C. are revivedby thawing at room temperature and 0.4 mL is transferred to 20 to 100 mLof medium, and the culture is incubated in a microaerobic environment at37° C. for 16 to 96 hours until the optical density (measured at 600 nm)is in a range from about 0.4 to about 20. This seed culture is then usedto inoculate a larger culture having a volume from about 200 mL to about2000 mL which is then incubated under the same conditions to achieve thesame cellular concentration as before. The larger culture is then usedto inoculate a small bioreactor having an operating volume of 2 L (e.g.,Biostat B, Sartorius-Stedim, Germany) or 10 L (e.g., Biostat C10,Sartorius-Stedim, Germany) or 16 L (e.g., New Brunswick Bioflo 510,Eppendorf, USA) or 50 L (e.g., Biostat D50, Sartorius-Stedim, Germany).The bioreactor is operated at 37° C. with pH, dissolved oxygen, and foamcontrol. The pH is controlled at a set point in the range from pH6 to pH8 such as by automatic addition of 10% (v/v) phosphoric acid or 10%(v/v) ammonia solution. Preferably, the bioreactor is sparged with a gasmixture containing nitrogen, carbon dioxide and a small proportion ofoxygen (or compressed air) and a dissolved oxygen saturation iscontrolled e.g., in a range from 0.5% to 10% saturation, such as byvarying stirrer speed and/or gas flow rate and/or vessel back pressure.Foam may be controlled by automatic addition of chemical antifoam, e.g.,polypropylene glycol, added as required.

In an example of production-scale bioreactor process, a bioreactorhaving a volume from about 400 L to about 10,000 L is operated in aconfiguration that enables high yield of H. pylori cells. The system maybe configured with an automated sterilization process and a series ofre-sterilizable sample(s) and addition valves, to enable sampling andaddition of reagent and product during fermentation. At inoculation, theinoculum is transferred aseptically to the production bioreactor. Thebioreactor is operated at 37° C. with pH, dissolved oxygen and foamcontrol, essentially as during the pilot-scale production process.

Fed-batch or “semi-batch” culture is particularly preferred forlarge-scale production of H. pylori. In fed-batch culture, one or morenutrients are fed to the bioreactor during cultivation and the cellularproduct remains in the bioreactor until the end of the run. In somecases, all the nutrients are fed into the bioreactor. Fed-batch culturepermits better control of the nutrient concentrations in the cultureliquid. Fed-batch H. pylori cultures are generally monitored for one ormore of dissolved oxygen concentration, feed composition to increasecell number, feed rate to increase cell number, gas requirement requiredto increase cell number, and nutrient composition of medium required toincrease cell number. This is because of the high nutrient demand of H.pylori. Optical density is monitored for comparative analysis of themedia formulations and cell growth, such that a high optical density ofcells is obtained in the shortest time frame. For example, a cellconcentration above levels typically observed in batch culture may beobtained, such as greater than 20 optical density units at 600 nm and/orup to about 40 optical density units at 600 nm.

3. Inactivating and Killing H. pylori

H. pylori may be inactivated and/or killed by chemical means and/orphysical means and/or genetic means. As used herein, the term “chemicalmeans” refers to a method of inactivating and/or killing H. pylori byexposing H. pylori to a chemical agent. As used herein, the term“physical means” refers to a method of inactivating and/or killing H.pylori by exposing H. pylori to one or more physical treatments notinvolving the use of a chemical. As used herein, the term “geneticmeans” refers to a method of inactivating and/or killing H. pylori bymodifying the genome of H. pylori.

Suitable chemical means for inactivating and/or killing H. pyloriinclude the addition of one or more chemical agents such as formaldehydeand/or β-propiolactone and/or ethyleneimine and/or binary ethyleneimineand/or thimerosal and/or acid and/or alkali and/or one or morebactericidal agents and/or one or more reducing agents and/or a bilesalt. Derivatives of these chemical agents known in the art may also beemployed.

In one preferred example, H. pylori is inactivated and/or killed byexposure to formaldehyde at a concentration from about 0.01% to about 1%(w/w) or from about 0.01% to about 0.1% (w/w) or between about 0.025%and about 0.1% (w/w).

Alternatively, or in addition, H. pylori is inactivated and/or killed byexposure to polyethyleneimine functionalized zinc oxide nanoparticles asdescribed, for example, by Chakraborti et al. 2012 Langmuir,28:11142-11152.

Alternatively, or in addition, killed H. pylori as described accordingto any example hereof is prepared by exposing live and/or inactivated H.pylori cells or strains to one or more bactericidal agent(s). Forexample, live and/or inactivated H. pylori can be subjected to treatmentwith one or more antibiotics selected from rifampin, amoxicillin,clarithromycin, rifamycin, rifaximin, the rifamycin derivative3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin syn.KRM-1648 and/or the rifamycin derivative3′-hydroxy-5′-(4-propyl-1-piperazinyl)benzoxazinorifamycin syn.KRM-1657.

Alternatively, or in addition, inactivated H. pylori as describedaccording to any example hereof is prepared by exposing live H. pyloricells or strains to one or more acid(s) or to a low pH environment suchas pH 3.0 or lower and/or to one or more base(s) or to high pHenvironment such as pH 9.0 or higher.

Alternatively, or in addition, inactivated and/or killed H. pylori asdescribed according to any example hereof is prepared by exposing liveH. pylori cells or strains to one or more reducing agent(s) such assodium bisulfite and/or one or more oxidative agents such as hydrogenperoxide.

Alternatively, or in addition, inactivated and/or killed H. pylori asdescribed according to any example hereof is prepared by exposing liveH. pylori cells or strains to bile salts.

Suitable physical means for inactivating and/or killing H, pyloriinclude exposure to visible light and/or ultraviolet light such as UV-Clight and/or low-power laser photosensitizer and/or heat (e.g., dry heator wet heat such as in steam) and/or elevated pressure and/ortemperature shift and/or freeze-thaw and/or freeze-drying(lyophilization) and/or sonication.

Alternatively, or in addition H. pylori is inactivated and/or killed byexposure to visible light at wavelengths ranging from about 375 nm toabout 500 nm or in a range from about 400 nm to about 420 nm.

Alternatively, or in addition, H pylori is inactivated and/or killed byexposure to ultraviolet light, e.g., Hayes el al. 2006, Appl. Environ.Microbiol. 72: 3763-3765.

For example, inactivated H. pylori as described according to any examplehereof is prepared by exposing live H. pylori cells or strains toirradiation such as ultraviolet irradiation and/or by exposure tovisible light such as wavelengths ranging from about 375 nm to about 500nm or in a range from about 400 nm to about 420 nm e.g., 405 nm violetlight. In one example, inactivated H pylori as described according toany example hereof is prepared by a process comprising exposing live Hpylori cells or strains to ultraviolet C (UVC) irradiation such aswavelength in a range from about 100 nm to about 280 nm such as about257.3 nm and/or to ultraviolet B (UVB) irradiation such as wavelength ina range from about 280 nm to about 315 nm and/or to ultraviolet A (UVA)irradiation such as wavelength in a range from about 315 nm to about 400nm. Preferably, the live H. pylori is exposed to UVC light in a rangefrom about 100 nm to about 280 nm such as about 257.3 nm and/or the liveH. pylori is exposed to about 405 nm violet light to thereby inactivateH. pylori.

Alternatively, killed H. pylori as described according to any examplehereof is prepared by a process comprising exposing live or inactivatedH. pylori cells or strains to ultraviolet C (UV-C) irradiation such aswavelength in a range from about 100 nm to about 280 nm e.g., about257.3 nm and/or to ultraviolet B (UV-B) irradiation such as wavelengthin a range from about 280 nm to about 315 nm and/or to ultraviolet A(UV-A) irradiation such as wavelength in a range from about 315 nm toabout 400 nm. Preferably, the live or inactivated H. pylori is exposedto UV-C light in a range from about 100 nm to about 280 nm such as about257.3 nm. Alternatively, the live or inactivated is exposed to about 405nm violet light to thereby kill H. pylori.

Alternatively, or in addition H. pylori is inactivated and/or killed byexposure to gamma irradiation.

Alternatively, or in addition, H. pylori is inactivated and/or killed byexposing live or inactivated H. pylori to low-power laser light in thepresence of a photosensitiser as described, for example, by MILLSON etal. 1996 J. Med. Microbiology, 44:245-252. Alternatively, or inaddition, H. pylori is inactivated and/or killed by heat treatment ofcells.

For example, H. pylori may be inactivated by heat treatment wherein liveH. pylori cells are exposed to heat treatment such as at temperatures inthe range between about 40° C. to about 70° C. or more. Preferred heattreatment in this context may comprise exposure of live H. pylori cellsto a temperature of about 60° C. or more for at least about 60 seconds,preferably at a temperature of about 60° C. or about 70° C. or about 80°C. or about 90° C. or about 100° C. or about 110° C. or about 120° C. orabout 130° C. or about 140° C. or about 150° C., said temperatureexposure being for a period of at least 3 minutes or at least 4 minutesor at least 5 minutes or at least 6 minutes or at least 7 minutes or atleast 8 minutes or at least 9 minutes or at least 10 minutes or at least20 minutes or at least 30 minutes or at least 40 minutes or at least 50minutes or at least 1 hour or at least 2 hours or at least 3 hours or atleast 4 hours or at least 5 hours or at least 6 hours or at least 7hours or at least 8 hours or at least 9 hours or at least 10 hours or atleast 11 hours or at least 12 hours or at least 13 hours or at least 14hours or at least 15 hours or at least 16 hours or at least 17 hours orat least 18 hours or at least 19 hours or at least 20 hours or at least21 hours or at least 22 hours or at least 23 hours or at least 1 day orat least 2 days or at least 3 days or at least 5 days or at least 5 daysor at least 6 days or at least 7 days.

Alternatively, killed H. pylori as described according to any examplehereof is prepared by exposing live and/or inactivated H. pylori cellsor strains to heat treatment such as by exposure to temperature of about60° C. or more for at least about 60 seconds, preferably at atemperature of about 60° C. or about 70° C. or about 80° C. or about 90°C. or about 100° C. or about 110° C. or about 120° C. or about 130° C.or about 140° C. or about 150° C., said temperature exposure being for aperiod of at least 2 minutes or at least 3 minutes or at least 4 minutesor at least 5 minutes or at least 6 minutes or at least 7 minutes or atleast 8 minutes or at least 9 minutes or at least 10 minutes or at least20 minutes or at least 30 minutes or at least 40 minutes or at least 50minutes or at least 1 hour or at least 2 hours or at least 3 hours or atleast 4 hours or at least 5 hours or at least 6 hours or at least 7hours or at least 8 hours or at least 9 hours or at least 10 hours or atleast 11 hours or at least 12 hours or at least 13 hours or at least 14hours or at least 15 hours or at least 16 hours or at least 17 hours orat least 18 hours or at least 19 hours or at least 20 hours or at least21 hours or at least 22 hours or at least 23 hours or at least 1 day orat least 2 days or at least 3 days or at least 5 days or at least 5 daysor at least 6 days or at least 7 days.

In one preferred example, live and/or inactivated H. pylori is killed byexposure to a single such elevated temperature or by exposure to atleast two different elevated temperatures such as by exposure to a firsttemperature of about 70° C. followed exposure to a second temperature ofabout 90° C. or about 95° C. In one such preferred example, the liveand/or inactivated H. pylori is killed by exposure to temperature ofabout 70° C. for about 10 minutes followed by exposure to temperature ofabout 90° C. or about 94° C. or about 95° C. for about 5 minutes.

Alternatively, or in addition, killed H. pylori as described accordingto any example hereof is prepared by exposing live and/or inactivated H.pylori cells or strains to elevated temperatures in the presence ofsteam and elevated pressure, such as by autoclaving live and/orinactivated H. pylori cells or strains. For example, live and/orinactivated H. pylori is killed by autoclaving the bacterial cells orstrains for about 15 minutes at about 121° C. and about 15 psi, or forabout 3 minutes at about at 132° C. and about 30 psi.

In one preferred example, H. pylori is inactivated and/or killed bytemperature shift such as exposure to a single such elevated temperatureor by exposure to at least two different elevated temperatures such asby exposure to a first temperature of about 70° C., followed exposure toa second temperature of about 90° C. or about 94° C. or about 95° C.

Alternatively, or in addition, H. pylori is inactivated and/or killedexposure of cells to one or more freeze-thaw cycles e.g., by exposure to2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 freeze-thaw cycles. Exemplaryfreeze-thaw cycles comprise freezing H. pylori in a dry ice/ethanol bathand then thawing the material at 37° C.

Alternatively, or in addition, H. pylori is inactivated and/or killed byfreeze-drying cells.

Alternatively, or in addition, H. pylori is inactivated and/or killed bysonicating the cells. For example, killed H. pylori as describedaccording to any example hereof is prepared by sonication e.g., atultrasonic frequencies such as about 20 kHz or more of live and/orinactivated H. pylori.

Preferably, the inactivated and/or killed H. pylori as describedaccording to any example hereof is prepared by first by exposing live H.pylori cells or strains to irradiation such as gamma irradiation and/orultraviolet irradiation such as UV-C light and/or by exposure to visiblelight such as wavelengths ranging from about 375 nm to about 500 nm orin a range from about 400 nm to about 420 nm, to thereby inactivate H.pylori and then exposing the inactivated H. pylori cells or strains toheat treatment as described according to any example hereof to therebykill the inactivated H. pylori or render the inactivated H. pyloriirreversibly metabolically inactive.

For example, the inactivated H. pylori is then exposed to temperature ofabout 60° C. or more for at least about 60 seconds, preferably at atemperature of about 60° C. or about 70° C. or about 80° C. or about 90°C. or about 100° C. or about 110° C. or about 120° C. or about 130° C.or about 140° C. or about 150° C., said temperature exposure being for aperiod of at least 2 minutes or at least 3 minutes or at least 4 minutesor at least 5 minutes or at least 6 minutes or at least 7 minutes or atleast 8 minutes or at least 9 minutes or at least 10 minutes or at least20 minutes or at least 30 minutes or at least 40 minutes or at least 50minutes or at least 1 hour or at least 2 hours or at least 3 hours or atleast 4 hours or at least 5 hours or at least 6 hours or at least 7hours or at least 8 hours or at least 9 hours or at least 10 hours or atleast 11 hours or at least 12 hours or at least 13 hours or at least 14hours or at least 15 hours or at least 16 hours or at least 17 hours orat least 18 hours or at least 19 hours or at least 20 hours or at least21 hours or at least 22 hours or at least 23 hours or at least 1 day orat least 2 days or at least 3 days or at least 5 days or at least 5 daysor at least 6 days or at least 7 days. In one such example, theinactivated H. pylori is exposed to a single such elevated temperatureor to at least two different elevated temperatures such as by exposureto a first temperature of about 70° C. e.g., for about 10 minutes,followed by exposure to a second temperature of about 90° C. or about95° C. e.g., for about 5 minutes.

In one preferred example, the killed H. pylori as described according toany example hereof is prepared by first by exposing live H. pylori cellsor strains to ultraviolet irradiation such as UVC light e.g., at aboutas 257.3 nm to thereby inactivate H. pylori and then exposing theinactivated H. pylori cells or strains to heat treatment as describedaccording to any example hereof to thereby kill the inactivated H.pylori or render the inactivated H. pylori irreversibly metabolicallyinactive.

Accordingly, in one preferred example, the composition according to anyexample hereof comprises H. pylori that has been subjected to a processfor inactivating H. pylori by irradiation and a process for the killingthe inactivated H. pylori by heat treatment.

Alternatively, or in addition, H. pylori as described according to anyexample hereof is inactivated and/or killed by exposing live orinactivated H. pylori to anaerobic conditions e.g., by changing theatmosphere in which H. pylori is cultured from microaerobic to anaerobicenvironment for example to mimic the in vivo atmospheric conditionsduring the washout of H. pylori from the stomach to the lower gut (e.g.,small and/or large intestine). For example, live (such as freshly grown)H. pylori is inactivated by exposing (e.g., by growing or incubating)the bacterial cells to anaerobic conditions for about 1 day to about 5days or more, including for at least about 24 hours, or for at leastabout 48 hours or at least about 73 hours or at least about 96 hours orat least about 120 hours. In one such example, the live H. pylori cellsare inactivated by exposing the cells to anaerobic conditions and byheat treatment of the cells.

In another example, live or inactivated H. pylori as described accordingto any example hereof is killed by exposing (e.g., by incubation) thelive or inactivated bacterial cells to anaerobic conditions for about 1day to about 5 days or more, including for at least about 24 hours, orfor at least about 48 hours or at least about 73 hours or at least about96 hours or at least about 120 hours.

In one preferred example, the composition according to any examplehereof comprises H. pylori that has been subjected to a process forinactivating H. pylori by exposing (e.g., by growing or incubating) thebacterial cells to anaerobic conditions for about 1 day to about 5 daysor more, including for at least about 24 hours, or for at least about 48hours or at least about 73 hours or at least about 96 hours or at leastabout 120 hours, and a process for the killing the inactivated H. pyloriby heat treatment of the cells.

Suitable genetic means for producing inactivated H. pylori as describedaccording to any example hereof comprises mutagenesis of live H. pyloricells or strains to modify one or more genes the expression of whichis/are required for efficient colonization and/or maintenance of Hpylori in the stomach and intestinal mucosa of human subject. Forexample, such genes may be deleted by recombination or modified byinsertion of a transposon or other genetic element, or they may beinactivated by chemical mutagenesis. Such means are described in theart.

Inactivation and/or killing may be performed on H. pylori cells that arein a liquid, semisolid or solid form. In one example, H. pyloricultivated in a liquid may be inactivated and/or killed during alogarithmic phase of growth i.e., wherein cell numbers are increasingexponentially in culture or stationary phase of growth i.e., whereinviable cells in culture are post-logarithmic and not increasing innumber.

In a particularly preferred example, a pilot-scale culture or otherlarge-scale culture e.g., greater than 2 L or greater than 5 L orgreater than 10 L or about 100 L to about 400 L volume including 100 Lor 150 L or 200 L or 250 L or 300 L or 350 L or 400 L, or larger volumeculture, is treated in a steam-in-place bioreactor orsterilisable-in-place bioreactor e.g., having the same operating systemas a pilot-scale reactor described herein. In such bioreactors, theinactivation and/or killing process utilizes high temperature and highpressure to generate steam which is applied to the cells once they haveachieved a desired cell density, to thereby inactivate and/or kill thecells

In another example, a culture in a fed-batch process is subjected toultraviolet light e.g., UV-C irradiation, at an irradiance of greaterthan 100 Joules per OD unit at 600 nm, and the cells are thenheat-treated at a temperature in a range from 60° C. to about 120° C.including 121° C. for a time in a range from about 15 minutes to about 6hours.

The present invention also provides a master cell bank comprising thetreated H. pylori of the present invention prepared as described herein.For example, a master cell bank may comprise aliquots of the treated H.pylori cells e.g., 100 or 200 or 300 or 400 or 500 vials comprising thecellular product. Preferably, a master cell bank is stored frozen e.g.,at −80° C.

4. Harvesting Treated H. pylori Cells

Methods for harvesting microorganisms are well known in the art and aredescribed, for example, by Ausubel et al. (In: Current Protocols inMolecular Biology. Wiley Interscience, ISBN 047 150338, 1987) orSambrook et al. (In: Molecular Cloning: Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).As used herein, the term “harvesting” refers to a collection of H.pylori from medium upon, or in which, a population of H pylori has beencultivated. Suitable methods include, for example, centrifugation e.g.ultracentrifugation, or by filtration, e.g. ultrafiltration ormicrofiltration or deep filtration.

H pylori cells are generally harvested after inactivation and/orkilling.

In a preferred example, treated cells are recovered from culture e.g., abioreactor, by employing continuous centrifugation such as at acentrifugal force in a range from about 5,000×g to about 20,000×g.Preferably, the harvested cells are washed using a formulation buffere.g., a phosphate-buffered saline or other pharmaceutically-acceptableexcipient or diluent. The wash solution may be supplemented with dextranor an encapsulation formulations.

The cellular product is then packaged ready for storage or use.Preferably, the cells are lyophilized or spray-dried to generate a solidproduct suitable for long term storage.

Packs comprise one or more specifications of cellular product e.g., cellconcentration, buffer composition, liquid formulation, dry formulation,pack size, etc.

5. Determining or Identifying Inactivated and/or Killed H. pylori

A method to measure utility of H. pylori or cell thereof in thecompositions and/or methods as described in any example hereof includesany method that measures the ability of H. pylori or cell thereof toreplicate and/or colonize a gastric mucosa of a mammal and/or any methodthat measures the ability of H. pylori or cell thereof to adhere to thegastric mucosa or epithelial cells thereof and/or any method thatmeasure viability and/or metabolic activity of H. pylori e.g., followingstress and/or inactivation and/or killing treatment of H, pylori asdescribed according to any example hereof.

In one example, the ability of a H. pylori or cell thereof to replicateand colonize the gastric mucosa in a mammal is determined byquantification of viable bacteria such as by colony count. See e.g.,Drumm and Sheman 1991, J. Med. Microbiol 35:197-202.

For example, 0.5 ml of a sample measuring cell equivalent of opticaldensity of about 0.2 to about 5.0 or more at 660 nm stored H. pyloribacteria which has been cultured, and subjected to stress and/orinactivation and/or killing treatment e.g., as described according toany example hereof, is resuspended in a 250 ml Erlenmeyer flaskcontaining 11 ml of fresh medium of Brucella Broth (e.g., GibcoLaboratories, Madison, Wis., USA) supplemented with 10% fetal bovineserum (e.g., Boknek Laboratories, Ontario, Canada), or other mediasuitable for growth of H. pylori e.g., as described according to anyexample hereof. If required, the culture medium is supplemented withtrimethoprim (e.g., Sigma Chemicals, St. Louis, Mo., USA) 5 mg/L and/orvancomycin (e.g., Sigma) 10 mg/L. The flask is closed with a looselyfitted screw cap and placed inside an incubation jar which is thenevacuated and flushed through with a gas mixture containing CO₂10%, O₂5%, N₂ 85%, and incubated at 37° C. on a rotary shaker and incubated at37° C. with shaking at 100 rpm. After about 24 hours, approximately 1 mlof bacterial culture is transferred to fresh medium of Brucella Brothand re-cultured. Absence of viable and replicating H. pylori isconfirmed by subculture of broth on supplemented Brucella agar platesand inspection of bacterial morphology by phase contract microscopy. Seee.g., Drumm and Sherman, 1989, J. Clin Microbiol, 27:1655-1656.

Alternatively, or in addition, quantification of viable and replicatingH. pylori bacteria is performed in the broth cultures by serial dilutionand sequential measurements of optical density of cultures at 600 nmand/or by colony counts e.g., after incubation for 6, 12, 18, 24, 30 and36 hours. For example, if present, cell counts of viable and replicatingH. pylori bacteria are determined by inoculating serial dilutions ofcultures in triplicates onto Brucella agar and incubating plates for 5days at 37° C. in microaerobic conditions. Viable and replicating H.pylori produce smooth, translucent colonies on Brucella agar. To improvethe accuracy of viable counts (cfu), 4 mg/L tetrazolium salts are addedto the Brucella agar prior to inoculation of the agar with the bacterialcultures. After incubation for 5 days at 37° C. in microaerobicconditions, viable and replicating H. pylori cells produce red colonieson this medium. Absence of H. pylori colonies as described hereinconfirms that H. pylori is inactivated and/or killed and is incapable ofreplicating and colonising the gastric mucosa.

In another example, inactivated and/or killed H. pylori may be confirmedby any assay measuring metabolic activity of H. pylori such as, forexample, urease production and/or ATP consumption.

In one preferred example, Rapid Urease Test, also known asCampylobacter-like organism (CLO) test, is used to detect presence of H.pylori which is partially or fully metabolically active based on theability of H. pylori to secrete a urease enzyme which catalyzes theconversion of urea to ammonia and CO₂. According to this example,aliquots of about 1 μl to about 100 μl, of samples measuring cellequivalent of optical density of about 0.2 to about 5.0 or more at 660nm of stored H. pylori bacteria cultured, and subjected to stress and/orinactivation and/or killing treatment e.g., as described according toany example hereof, or aliquots of about 1 μl to 100 μl of bacterialcells cultured in any suitable medium e.g., Brucella Broth medium asdescribed above, are added to sterile Eppendorf tubes containing freshlyprepared urease indicator reagent to a total volume of 200 μl. Forexample, a urease indicator reagent containing about 2% (w/v) to about5% (w/v) urea, and at least one pH indicator such as phenol red,bromothymol blue, bromocresol purple, and methyl red at a concentrationof about 0.1% (w/v) or about 0.05% (w/v) in 0.01 M phosphate-bufferedsaline (PBS), may be used. If required, the pH of each urease indicatorreagent formulation may be adjusted to the lower end of the known pHrange for each indicator with the use of 0.1 N or 1.0 N HCl; Forexample, the indicator phenol red has a pH range of 6.6 to 8.0 and aurease indicator reagent formulation comprising phenol red may beadjusted to pH 6.6; the indicator bromothymol blue has a pH range of 6.0to 7.6 and a urease indicator reagent formulation comprising bromothymolblue may be adjusted to pH 6.0; the indicator the indicator bromocresolpurple has a pH range of 5.2 to 6.8 and a urease indicator reagentformulation comprising bromocresol purple may be adjusted to pH 5.2; theindicator the indicator Methyl red has a pH range of 4.8 to 6.2 and aurease indicator reagent formulation comprising bromocresol purple maybe adjusted to pH 4.8. Alternatively, the urease indicator reagent isprepared as described by Nedrud J G, Blanchard T G. Helicobacter animalmodels. In: Coligan J E, Bierer B, Margulies D H, Shevach E M, StroberW, Coico R, editors. Current Protocols in Immunology. Philadelphia: JohnWiley and Sons; 2000. p. 19.8.1-26. Alternatively, the urease indicatorreagent may be obtained commercially e.g., from ASAN pharm. Co., Seoul,Korea. The tubes are then vortexes and incubated at room temperature.After 4 hours the tubes are centrifuged at RCF 6000 for 5 minutes andabout 100 μl of the supernatant is transferred to a 96-well plate to beread spectrophotometrically at 550 nm. If required, Gastric mucosaltissue homogenates from mice uninfected with H. pylori e.g., prepared asdescribed below may serve as negative control for the urease assay.Also, if required, a positive control containing known concentrations ofcultured H. pylori such as wild type H. pylori capable of replicatingand colonizing the mucosa may be used. Samples of H. pylori showing lessthan 5% urease activity as determined by the Rapid Urease Test indicateH. pylori which is inactivated and/or killed.

As will be apparent to the skilled artisan, a number of urease tests arecommercially available, such as, for example, CLOtest (Kimberly-Clark),Hpfast (Sigma) and Pyloritek (Serim).

In another example, inactivation and/or killing of H. pylori isconfirmed by performing an oxidase test. As used herein, the term“oxidase test” shall refer to an assay used to detects the presence of acytochrome c oxidase using a redox indicator such as, for example,N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) orN,N-dimethyl-p-phenylenediamine (DMPD). Suitable oxidase tests aredescribed for example by Tsukita et al. 1999 J. Biochem. 1235:194-201 orMurray et al. (In: Manual of Clinical Microbiology, American Society forMicrobiology, Washington D.C., Ninth Edition 2007).

In another example, inactivation and/or killing of H. pylori isconfirmed by performing a catalase test. The term “catalase test” shallbe taken to encompass any assay that determines the ability of H. pylorito liberate oxygen gas from hydrogen peroxide by catalase degradation.Suitable catalase test will be apparent to the skilled artisan.

In yet another example, inactivation and/or killing of H. pylori isconfirmed by performing a motility assay as described, for example, byWorku et al. 1999 Microbiology 145: 2803-2811.

In another example, the ability of a H. pylori or cell thereof toreplicate and/or colonize the gastric mucosa of a mammal is determinedusing in vitro assay of H. pylori adherence to human gastric tissue. Seee.g., Hemalatha et al. 1991, J. Med. Microbiol 35:197-202; Falk et al.,1993, Proc. Natl. acad. Sci. USA. 90:2035-2039; Hsieh et al. 2012Helicobacter 17:466-477.

In another example, the ability of a H. pylori or cell thereof toreplicate and colonize the gastric mucosa in a mammal is determined byanalysis of in vivo stomach colonization infected animals e.g., mice.For example, H. pylori bacteria which has been cultured, and subjectedto stress and/or inactivation and/or killing treatment e.g., asdescribed according to any example hereof, is harvested and resuspendedin sterile saline. As a positive control a culture of H. pylori known tobe capable of replicating and colonizing the gastric mucosa of a mammaland which has not been the subjected to stressing and/or inactivationand/or killing treatment prior to inoculation challenge described below,is used. Suitable H. pylori capable of replicating and colonizing thegastric mucosa are known in the art. Briefly, a flask containing BHIbroth plus 4% fetal calf serum (FCS) is inoculate with an aliquot of apositive control H. pylori stock and allowed to incubate for 25 to 48hours at 37° C. in an atmosphere of 10% CO₂+5% O₂, shaking at 125 rpm,to yield a pure culture of H. pylori bacteria having the expectedmorphology to be used for infection. For challenge inoculum an opticaldensity of a 1:10 dilution of the sterile saline suspension comprisingthe H. pylori bacteria which has been cultured, and subjected to stressand/or inactivation and/or killing treatment is read at 660 nm, andinoculum samples generating a reading of between 0.07 and 0.002 are usedfor inoculation of mice.

Alternatively, samples for inoculation comprising an amount of H. pyloribacteria which has been cultured and subjected to stress and/orinactivation and/or killing treatment in a range equivalent to betweenabout 1×10⁷ to 2×10¹⁰ cells/ml or CFU/ml as determined e.g., by ahaemocytometer, are used. For positive control H. pylori, an inoculum ofabout 1×10⁸ cells is used. Six to 8 weeks old C57BL/6 mice (from CharlesRiver Laboratories (Wilmington, Mass.) and/or BALB/c (from Charles RiverLaboratories (Wilmington, Mass.) are challenges orally with a dosagecomprising an amount of bacteria in a range corresponding between 10⁸ to10¹⁰ cells, preferably 10⁹ cells of H. pylori bacteria subjected tostressing and/or inactivation and/or killing treatment and an inoculumof about 1×10⁸ cells comprising the control H. pylori, by gavage twicewithin a 1-week period, preferably at least one day separating eachchallenge.

Alternatively, mice are challenged by intragastric immunization whereinabout 0.25 ml or about 0.5 ml or about 1 ml volumes comprising inoculumdosages as above are delivered into the stomach of lightly etherizedmice by intubation through polyethylene tubing attached to a hypodermicsyringe. If required, this procedure may be performed three times in a5-day period, with 24 hours between dosing.

According to this example, for the purpose of analysing stomachcolonization two weeks following challenge mice are sacrificed e.g., byCO₂ inhalation and stomachs and duodenum are removed for quantitativeassessment of colonization. For example, the stomachs and duodenum aretransferred to labelled sterile Petri plates containing 5-10 ml ofsterile PBS, and are then transferred to a biosafety cabinet where thestomachs are opened by midline incision and the contents gently cleanedusing sterile gauze. The antrum is visualized and aseptically dissectedaway from the rest of the stomach, which is discarded. The antralsection is then diced, using sterilized single-edge razor blades, andthe pieces placed in a pre-weighed 5 ml tube containing brain-heartinfusion broth (BHI) media. If required, tubes containing antralsections are re-weighed to 0.001 g accuracy and placed in a biosafetycabinet. The sections may then be mechanically macerated e.g., usingsterile plastic tissue homogenizers and serial 1:10, 1:100, and 1:1000dilutions of the homogenates are made in BHI media. From each dilutiontube a 100 μl aliquot is placed on a sterile BHI agar plate and a fullplate spread is performed. For example, the media on which homogenatesare plated contain BHI agar (Difco), 4% fetal bovine serum, bacitracin,nalidixic acid, amphoteracin B, and Campylobacter selective supplement(Oxoid, Lenexa, Kans.). Plates are placed in anaerobic jars containingBBL CampyPak Plus microaerophilic envelopes (Becton Dickinson, FranklinLakes, N.J.; product #271045) and preferably incubated at 37° C. for 6-7days. Growth control plates are included in each jar, inoculated with afreshly grown preparation of the positive control H. pylori supra. Thelimit of detection in this assay is approximately 500 H. pylori cellsper gram of stomach tissue. Absence of H. pylori colonies indicates thatthe H. pylori is inactive and is unable to replicate and colonise thegastric mucosa. However, if colonies are observed on plates, coloniesmay be confirmed to be H. pylori by means as described in any examplehereof e.g., using one urease activity assay and/or oxidase activityassay and/or catalase activity assay and/or by colony morphology.

In yet another example, the ability of a H. pylori or cell thereof toreplicate and colonize the gastric mucosa in a mammal is determined bypolymerase chain reaction (PCR) detection of colonization of H. pyloriin conventional euthymic mice based on detection of the H. pylori 16Sribosomal gene sequence. See e.g. Smith et al., 1996, Clinic. Diagn.Lab. Immuno. 3:66-72. For example, H. pylori bacteria which has beencultured, and subjected to stress and/or inactivation and/or killingtreatment e.g., as described according to any example hereof, isharvested and resuspended in sterile saline. If required, as a positivecontrol a culture of H. pylori e.g., wild type (WT) H. pylori, known tobe capable of replicating and colonizing the gastric mucosa of a mammaland which has not been the subjected to stressing and/or inactivationand/or killing treatment prior to inoculation challenge described below,is separately harvested and resuspended in sterile saline. Suitable H.pylori capable of replicating and colonizing the gastric mucosa areknown in the art and are described herein. For challenge inoculum anoptical density of a 1:10 dilution of the inoculum is read at 660 nm,and inoculum samples generating a reading of between 0.07 and 0.002 areused for inoculation of mice.

Alternatively, inoculum samples comprising an amount of bacteria in arange corresponding to between about 2×10⁷ to 2×10⁸ cells/ml or CFU/mlas determined for example by a haemocytometer, are used. Eight to 12weeks old VAF and GF Swiss-Webster mice or VAF CD-1 mice (from TaconicLaboratories (Germantown, N.Y.)) and/or VAF-CD-1 mice (from CharlesRiver Laboratories (Wilmington, Mass.) and/or C57BL/6 mice (from CharlesRiver Laboratories (Wilmington, Mass.) and/or BALB/c (from Charles RiverLaboratories (Wilmington, Mass.) and/or SJL/J mice (from The JacksonLaboratory, Bar Harbor, Me., USA) are challenges orally with 0.5 ml ofthe H. pylori inoculum prepared as described above, by gavage twicewithin a 1-week period, preferably at least one day separating eachchallenge. All mice are housed in sterile microisolator cages withsterile water and mouse chow ad libitum. If required GF mice aremaintained and manipulated using sterile GF procedure, in laminar flowhood with all surfaces sanitized, and cages for GF mice are autoclavedin sterile wrap, and water is also autoclaved and filter sterilizedprior to use. To assess H. pylori colonisation of the gastric mucosa inchallenged mice, about 1 to about 4 weeks post challenge, preferablyabout 4 weeks post challenge, mice are sacrificed e.g., by inhalation ofCO₂ and stomachs are removed by aseptic techniques. Stomachs are thencut longitudinally, and the stomach contents are washed away by rinsingwith sterile deionized H₂O. The stomach mucosa is then separated fromthe stomach lining tissue by gently scraping the mucosa with sterileglass microscope slides. Mucosa samples are then placed in Tris-sodiumchloride-EDTA (TNE) buffer and stored on ice or frozen until DNAextraction for PCR analysis. Methods for extracting DNA from the mucosasuspensions for PCR analysis are known in the art and may be readilyemployed. For example, DNA are extracted by centrifuging the stomachmucosa suspension in TNE buffer for 3 min at 12,000 rpm. The supernatantis then removed, and the cell pellet is resuspended in 570 ml of TNEcontaining 1% Triton X-100 (Sigma) and 0.5 mg of lysozyme (Sigma) perml. Samples are then incubated at 37° C. for 30 min. Next, 1 mg ofproteinase K (Boehringer GmbH, Mannheim, Germany) per ml is added, andthe mixture is incubated at 65° C. for 2 hours or at 37° C. overnight.The digest is mixed with an equal volume of phenol-chloroform-isoamylalcohol (25:24:1) and then centrifuged at 10,000 3 g for 6 min. The topaqueous layer is then removed, and a second extraction withphenol-chloroform-isoamyl alcohol is preferably performed. The aqueouslayer is then mixed with an equal volume of chloroform-isoamyl alcohol(24:1) and processed as in the previous two extractions. DNA is thenprecipitated by adding a 1/10 volume of 3 M sodium acetate and 2 volumesof absolute ethanol and placing on dry ice for 20 min. DNA is pelletedby centrifugation as described above and rinsed with 70% ethanol. Thepellet is preferably dried e.g., by speed vacuum and resuspended in 100ml of 0.13 TE (13 TE is 10 mM Tris [pH 7.4], 0.1 mM EDTA). Samples arestored at 4° C. until the PCR is run.

PCR primers used for amplification of a DNA sequence of H. pyloriencoding the 16S rRNA which are used include the upstream primer (HPforward) set forth in SEQ ID NO: 1 had having the sequence 5′-TTG GAGGGC TTA GTC TCT-3′, and the downstream primer (HP reverse) set forth inSEQ ID NO: 2 and having the sequence 5′-AAG ATT GGC TCC ACT TCA CA-3′.The primers set forth in SEQ ID NO: 1 and SEQ ID NO: 2 are designed to459 bp PCR product spanning bases 793 to 1252 of the H. pylori DNAsequence. SEQ ID NO: 1 and SEQ ID NO: 2 primers are designed based on aregion of homology for six isolates of H. pylori listed in gene bankaccession numbers U00679, U01328, U01329, U01330, U01331, and U01332 andwhich differs from the sequences listed for H. felis (gene bankaccession number M57398), H. muridarum (gene bank accession numberM80205), and Campylobacter sp. (gene bank accession number L04315).Accordingly use of these primers avoids cross-reactivity with closelyrelated bacteria.

If required, internal PCT control DNA templates can also be constructedfor use in the PCR reaction. See e.g, Smith et al, 1996 supra. Forexample, the 495-bp PCR product amplified from a WT H. pylori using theprimers set forth in SEQ ID NO 1 and SEQ ID NO: 2 as described accordingto any example hereof, is closed into a multi-copy plasmid.Subsequently, an internal restriction fragment of 237 bp, convenientlyflanked by SM sites, is deleted from within the cloned H. pylori DNA tocreate a template with perfect homology to the HP primers but from whicha much shorter sequence would be amplified with those primers. The459-bp PCR amplified H. pylori DNA fragment was purified by using aGENECLEAN® kit (Bio 101, Inc., La Jolla, Calif.) 15 and ligated with T4DNA ligase (GIBCO BRL, Gaithersburg, Md.) into the EcoRV site of plasmidpBLUESCRIPT II SKI® (Stratagene Co., La Jolla, Calif.), which confersampicillin resistance and encodes the lacZa peptide. The recombinantplasmids are transformed into Escherichia coli DH5a (GIBCO BRL).Ampicillin-resistant transformants are selected on Luria broth platescontaining X-Gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside), andplasmids carrying inserted DNA are identified as giving white colonies.Plasmids are extracted from selected colonies with the Qiagen plasmidpurification kit (Qiagen Inc., Chatsworth, Calif.) and cut with Stylrestriction endonuclease (New England BioLabs, Beverly, Mass.), whichremoves an internal 237-bp DNA fragment from the H. pylori DNAinsertion. The remaining DNA is recircularized with T4 DNA ligase andtransformed into strain DH5a, and colonies are selected as ampicillinresistant. Transformants yield the desired 222-bp fragment whenamplified in PCRs with the HP primers set forth in SEQ ID NO: 1 and SEQID NO: 2. One transformant may be selected was selected, and plasmid DNAis extracted for use as the internal PCT control template.

PCT analysis is conducted by preparing master reaction mixtures, understerile conditions. Each master mixture is made in in a 1.5-mlmicrocentrifuge tube and contains reactants for 45 sample reactions. Toeach master mix there is added 826.9 ml of deionized H₂O, 112.5 ml of103 Taq buffer (Stratagene), 45 ml of deoxynucleoside triphosphate (5mM), 22.5 ml of each primer of SEQ ID NO: 1 or SEQ ID N: 2 (25 to 50mM), and 5.6 ml of Taq polymerase (5 U/ml; Stratagene). If required, themaster mixtures are aliquoted at 23 ml per reaction into 200-ml PCRtubes. The volume of each reaction mixture for PCR is brought up to 25ml by adding 2 ml of DNA templates extracted mouse mucosal DNA extractedabove, typically comprising an amount of 2 mg of extracted DNA. Ifrequired, PCR reaction tubes are briefly centrifuged to mix reactants.PCR reaction mixtures containing the mucosal DNA extracted from micechallenged with H. pylori subject to stressing and/or inactivationand/or killing treatment and, if required, from negative control micechallenges with WT H. pylori are cycled in Perkin-Elmer 9600 Systemthermal cycler (Perkin-Elmer, Norwalk, Conn.). DNA is amplified for 35cycles of 15 s at 94° C., 30 s at 55° C., and 1 min at 72° C., with afinal elongation cycle at 72° C. for 10 min. Positive and negativecontrol reactions may be perfumed performed with each amplification. Ifrequired, control templates in each PCR run may be used which consist ofdeionized H₂O, H. pylori DNA corresponding to 1, 10, and 100 cells, andmouse mucosal tissue DNA (2 mg). The PCR products are analyzed by 2%agarose gel electrophoresis with ethidium bromide incorporation andvisualized under UV light. Detection of a PCR product is scored ascolonization, while absence of a PCR product is scored asno-colonization, and provides a positive confirmation that the H. pyloriis inactive and is unable to replicate and colonise the gastric mucosaof a mammal.

Other methods for measuring the utility of H. pylori or cell thereof inthe compositions and/or methods as described in any example hereof willbe apparent to the skilled artisan and are encompassed by the presentinvention.

6. Formulations

Inactivated and/or Killed H. pylori or Cell Lysates Thereof May beFormulated for Oral Administration to a Human or Mammal.

In one example, inactivated and/or killed H. pylori or a cell lysatethereof is encapsulated. As used herein, the term “encapsulated” shallbe taken to mean that the inactivated and/or killed H. pylori or celllysate is enclosed within a degradable barrier.

For example, the degradable barrier may degrade at a predeterminedlocation in gastrointestinal tract.

In one example the composition is in the form of a tablet or a capsule.

In another example, composition of the present invention is lyophilised.In another example, the composition of the present invention is apowder.

Compositions of the present invention may be formulated as a foodstuffor dietary supplement. As used herein, the term “foodstuff” refers toany food product or beverage and the term “dietary supplement” refers toa product intended to supplement the diet of a human or mammal thatcomprises a vitamins and/or a mineral and/or a herb or other botanicaland/or an acid.

In one example, the foodstuff or dietary supplement may be aready-to-drink product. As used herein, the term “ready-to-drink” shallbe taken to mean that the product is in a form suitable for oraladministration without additional preparation. Suitable ready-to-drinkproducts may include, for example, carbonated water, flavoured water,carbonated flavoured water, drinks containing juice (juice derived fromany fruit or any combination of fruits, juice derived from any vegetableor any combination of vegetables), milk drinks obtained from animals,milk drinks derived from soy, rice, coconut or other plant material,yoghurt drinks, sports drinks, energy drinks, coffee, decaffeinatedcoffee, tea, tea derived from fruit products, tea derived from herbproducts, decaffeinated tea and liquid meal replacements. In oneexample, the ready-to-drink product may comprise filtered water, skimmilk powder, cane sugar, wheat maltodextrin, soy protein, vegetableoils, starch, inulin, corn syrup solids, fructose, cereals, flavour,calcium, phosphorus, fermented red rice, vitamin C, Niacin, vitamin A,vitamin B12, vitamin B6, vitamin B2, vitamin BI, folate and salt. Inanother example, the ready-to-drink product may comprise includecarbonated water, corn syrup, caramel color, caffeine, phosphoric acid,coca extract, lime extract, vanilla and glycerine. In yet anotherexample, the ready-to-drink product may comprise carbonated water,sucrose, glucose, sodium citrate taurine, glucuronolactone, caffeine,inositol, niacinamide and vitamin B 12.

In another example, the foodstuff or dietary supplement may be aready-to-eat product. As used herein, the term “ready-to-eat” shall betaken to mean that the product is in a form suitable for oraladministration without additional preparation. Suitable ready-to-eatproducts may include, for example, a meal replacement bar, a proteinbar, snack food and confectionary product. In one example, theready-to-eat product may comprise wholegrain cereals, glucose, sugar,vegetable oil, maize starch, humectants, rice flour, oat flour, skimmilk powder and honey.

In yet another example, the foodstuff or dietary supplement may requiresuspension and/or reconstitution in a liquid or diluent prior toadministration. For example, the foodstuff or dietary supplement may bea liquid or liquid concentrate or powder.

In one example, the foodstuff or dietary supplement may be an infantformula or follow-on formula or infant formula for special dietary useor pre-term formula. As used herein, the term “infant formula” shallrefer to a breast milk substitute which satisfies the nutritionalrequirement of infants aged up to about four to about six months. In oneexample, the infant formula may have an energy content of no less thanabout 2500 kJ/L and no more than about 3150 kJ/L. In one example, theinfant formula may comprise an amount of protein between 0.45 g per 100kJ and 0.7 g per 100 kJ, an amount fat between 1.05 g per 100 kJ and 1.5g per 100 kJ. In another example, the infant formula may comprise lessthan 0.05 mg of aluminium per 100 mL. As used herein, the term“follow-on formula” shall refer to a breast milk substitute or areplacement for infant formula which constitutes the principal liquidsource of nourishment for infants aged from about six months. Forexample, infant follow-on formula may have an energy content of no lessthan about 2500 kJ/L and no more than about 3550 kJ/L. In one example,the infant formula may comprise an amount of protein between 0.45 g per100 kJ and 1.3 g per 100 kJ, an amount fat between 1.05 g per 100 kJ and1.5 g per 100 kJ. In another example, the infant formula may compriseless than 0.05 mg of aluminium per 100 mL. The term “infant formulaproduct for special dietary use” as used herein shall be taken to meanan infant formula product formulated to satisfy particular needs ofinfants with a particular metabolic and/or immunological and/or renaland/or hepatic and/or malabsorptive condition. For example, infantformula products for specific dietary use may have an energy content ofno less than about 2500 kJ/L and no more than about 3550 kJ/L. In oneexample, the infant formula may comprise an amount of protein between0.45 g per 100 kJ and 1.3 g per 100 kJ, an amount fat between 0.93 g per100 kJ and 1.5 g per 100 kJ. In another example, the infant formula maycomprise less than 0.05 mg of aluminium per 100 mL. The term “pre-termformula” shall be construed broadly to mean an infant formula productspecifically formulated to satisfy particular needs of an infant bornprior to 36 weeks of gestation. Preferably, pre-term formula maycomprise an amount of protein between 0.45 g per 100 kJ and 1.3 g per100 kJ, an amount fat between 0.93 g per 100 kJ and 1.5 g per 100 kJ. Inanother example, the infant formula may comprise less than 0.02 mg ofaluminium per 100 mL.

Compositions of the invention may comprise one or more prebiotics orparaprobiotics or probiotics e.g., as a food, beverage, dietarysupplement or animal feed.

The term “probiotic” used herein shall be taken to mean livemicroorganisms, which when administered in an adequate amount confers ahealth benefit on the host. Suitable probiotics include, for example,Aspergillus niger, Aspergillus oryzae, Bacillus coagulans, Bacilluslentus, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis,Bacteroides amylophilus, Bacteroides capillosus, Bacteroides ruminocola,Bacteroides suis, Bifidobacterium adolescentis, Bifidobacteriumanimalis, Bifidobacterium bifidum, Bifidobacterium infantis,Bifidobacterium longum, Bifidobacterium thermophilum, Enterococcuscremoris, Enterococcus diacetylactis, Enterococcus faecium, Enterococcusintermedius, Enterococcus lactis, Enterococcus thermophilus,Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillusbulgaricus, Lactobacillus casei, Lactobacillus cellobiosus,Lactobacillus curvatus, Lactobacillus delbruekii, Lactobacillusfermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillusplantarum, Lactobacillus reuteri, Leuconostoc mesenteroides, Pediococcusacidilacticii, Pediococcus pentosaceus, Propionibacteriumfreudenreichii, Propionibacterium shermanii, Saccharomyces cerevisiae.

The term “paraprobiotic” has been coined to refer to those productscomprising killed or inactivated microbes which may positively affecthost health (Taverniti V and Guglielmetti S, 2011, Genes Nutr. 6(3):261-274).

As used herein, the term “prebiotic” shall be taken to mean anon-digestible food ingredient that beneficially affects a host byselectively stimulating growth and/or activity of one or moremicroorganisms in the gut. Suitable prebiotics include, for example,fructooligosaccharides, transgalactooligosaccharides, inulins, acaciagum, xylooligosaccharides, isomaltooligosaccharides, lactulose and soyoligosaccharides.

7. Administration

Compositions of the present invention may be formulated for daily orperiodic administration. For example, the composition may beadministered daily for a period of at least about 1 week or at leastabout 2 weeks or at least about 3 weeks or at least about 4 weeks or atleast about 5 weeks or at least about 6 weeks or at least about 7 weeksor at least about 8 weeks or at least about 9 weeks or at least about 10weeks or at least about 11 weeks or at least about 12 weeks or at leastabout 13 weeks or at least about 14 weeks or at least about 15 weeks orat least about 16 weeks or at least about 17 weeks or at least about 18weeks or at least about 19 weeks or at least about 20 weeks or at leastabout 21 weeks or at least about 22 weeks or at least about 23 weeks orat least about 24 weeks or at least about 25 weeks, or at least about 6months, or at least about one year or more than one year. Preferably,the composition is administered, over a period of at least about 13weeks or at least about 3 months.

In another example, the composition may be administered periodically,such as, for example, every second day or every third day or everyfourth day or every fifth day or every sixth day or every second weekfor a period of at least about 1 week or at least about 2 weeks or atleast about 3 weeks or at least about 4 weeks or at least about 5 weeksor at least about 6 weeks or at least about 7 weeks or at least about 8weeks or at least about 9 weeks or at least about 10 weeks or at leastabout 11 weeks or at least about 12 weeks or at least about 13 weeks orat least about 14 weeks or at least about 15 weeks or at least about 16weeks or at least about 17 weeks or at least about 18 weeks or at leastabout 19 weeks or at least about 20 weeks. In yet another example, thecomposition may be administered intermittently. For example, thecomposition may be administered for an administration period at leastabout 1 week or at least about 2 weeks or at least about 3 weeks or atleast about 4 weeks or at least about 5 weeks or at least about 6 weeksor at least about 7 weeks or at least about 8 weeks or at least about 9weeks or at least about 10 weeks or at least about 11 weeks or at leastabout 12 weeks or at least about 13 weeks or at least about 14 weeks orat least about 15 weeks or at least about 16 weeks or at least about 17weeks or at least about 18 weeks or at least about 19 weeks or at leastabout 20 weeks or at least about 21 weeks or at least about 22 weeks orat least about 23 weeks or at least about 24 weeks or at least about 25weeks, or at least about 6 months, followed by a period ofdiscontinuance, followed by an administration period at least 1 week orat least 2 weeks or at least 3 weeks or at least 4 weeks or at least 5weeks or at least 6 weeks or at least 7 weeks or at least 8 weeks or atleast 9 weeks or at least 10 weeks or at least 11 weeks or at least 12weeks or at least 13 weeks or at least 14 weeks or at least 15 weeks orat least 16 weeks or at least 17 weeks or at least 18 weeks or at least19 weeks or at least 20 weeks or at least about 21 weeks or at leastabout 22 weeks or at least about 23 weeks or at least about 24 weeks orat least about 25 weeks, or at least about 6 months. Preferably, whereinthe composition is administered for a period of at least about 13 weeksor at least about 3 months, followed by a period of discontinuance, andthen followed by an administration period of at least about 13 weeks orat least about 3 months. In another example, the composition may beadministered for an administration period of at least 1 or 2 or 3 or 4of 5 or 6 or 7 or 8 or 9 or 10 or 15 or 20 or 25 or 30 or 35 or 40years.

In one example, compositions may be formulated as a daily dosagecomprising H. pylori or cell lysate thereof in an amount correspondingto about 10⁶ cells or about 10⁷ cells or about 10⁸ cells or about 10⁹cells or about 10¹⁰ cells or about 10¹¹ cells or about 10¹² or betweenabout 10⁶ cells to about 10¹² cells or between about 10⁷ cells to about10¹¹ cells or between about 10⁸ cells to about 10¹⁰ cells or betweenabout 10⁹ cells to about 10¹⁰ cells. As will be apparent to the skilledartisan, single or multiple dosage units may be administered to make upthe daily dosage.

In another example, compositions may be formulated for administration toinfants aged between 0 to about 5 years, or between 0 to about 4 years,or between 0 to about 3 years, or between 0 to about 2 years, or between0 to about 1 year. In one example, the composition may be formulated foradministration to infants aged between 0 to about 2 years. In anotherexample, the composition may be formulated for administration to infantsof an age between about 4 months and about 12 months. In anotherexample, the composition may be formulated for administration to infantsless than about 6 months of age.

In yet another example, compositions may be formulated foradministration to children older than about 5 years of age and/or toadolescents and/or to adults.

In a further example, a composition of the invention according to anyexample hereof is a cosmetic or a nutraceutical formulation such as afood stuff, tablet, capsule, or liquid drink, for administration to asubject not suffering from a medical condition referred to herein suchas allergy or one or more of allergic eczema, urticaria, hives,rhinitis, wheezing, airway resistance, airway restriction, lunginflammation, food allergy, or asthma or in need of prevention of suchmedical condition. For example, a cosmetic or a nutraceuticalformulation of the present invention will promote a general sense ofwellbeing and/or boost the immune system and/or provide balance to theimmune system of a subject not in need of therapy or prophylaxis fromany medical condition(s).

In one example, the present invention provides a method of cosmetic ornutraceutical use comprising administering a composition comprisinginactivated and/or killed H. pylori or a cell lysate thereof accordingto any example hereof to a subject not suffering from a medicalcondition referred to herein such as allergy or one or more of allergiceczema, urticaria, hives, rhinitis, wheezing, airway resistance, airwayrestriction, lung inflammation, food allergy, or asthma or in need ofprevention of such medical condition. In one such example, the method ofthe present invention promotes a general sense of wellbeing and/or boostthe immune system and/or provide balance to the immune system of asubject not in need of therapy or prophylaxis from any medicalcondition(s).

The present invention is described further in the following non-limitingexamples:

Example 1 Treatment to Inactivate and/or Kill H. pylori Cells—Method I

This example demonstrates the utility of ultraviolet irradiation, andoptional additional freeze-thawing, for inactivating and/or killing H.pylori cells.

Cells of Helicobacter pylori strain OND79 deposited with the NationalMeasurement Institute (NMI) of Australia under Accession No. V13/023374were obtained by growth on Columbia agar (CBA) plates comprisingColumbia agar base (Product Code CM0311, Thermo Fisher Scientific, OxoidLtd) and 7% (v/v) sterile defibrinated horse blood for 24 hours andharvesting cells by resuspension of grown cells in saline solution [0.9%(w/v) sodium chloride] and then centrifugation, according to standardprocedures. The cells were then resuspended in saline solution, and theconcentration of resuspended cells was adjusted to a measured absorbanceat 600 nm wavelength of 1 optical density (OD) unit per ml. Equalvolumes (1 ml) of resuspended cells were plated onto CBA platescomprising 7% (v/v) sterile defibrinated horse blood. The plates wereincubated for 24 hours at 37° C. in a microaerobic environmentcontaining 5% (v/v) CO₂ and less than 5% (v/v) O₂.

Plate samples were then subjected to ultraviolet irradiation usingultraviolet C (UV-C) light (wavelength between 200 and 290 nm) inBio-Link BLX crosslinker UV chamber (Vilber Lourmat, France) at anirradiance of 4 Joules/cm² or 12 Joules/OD₆₀₀ of plated cells. Forexample, a plate of 9 cm diameter may be irradiated by exposure to about240 Joules UV-C. Irradiated bacteria were then collected from theplates, resuspended in the saline solution and the concentration ofresuspended cells was adjusted to a measured absorbance at 600 nmwavelength of 20 optical density (OD) unit per ml.

As untreated control, OND79 H. pylori cells which were cultured,harvested and plated as described above but which were not irradiatedusing UV-C were also collected and resuspended in a saline solution andthe concentration of untreated resuspended cells was adjusted to ameasured absorbance at 600 nm wavelength of 20 optical density (OD) unitper ml.

Aliquots of the irradiated cells were assayed directly to determine cellreplication ability and urease activity, or alternatively, frozen at−20° C. and then thawed, prior to cellular replication and ureasetesting being performed.

To test for an ability of irradiated cells, optionally subjected toirradiation and freeze-thawing, to replicate, the bacterial suspensionswere serially-diluted in saline, plated onto CBA plates, and the platesincubated for 3 days at 37° C. in a microaerobic environment containing5% (v/v) CO₂ and less than 5% (v/v) O₂. Cell counts were then determinedfor the various dilutions tested.

In two independent experiments, no colony forming units were identifiedfor suspensions in a concentration range corresponding to a measuredabsorbance at 600 nm wavelength of 0.056-3.6 OD units per ml. The sameresults were obtained for cells receiving only UV-C as for cellsreceiving UV-C and a cycle of freeze-thawing.

Urease activities of the treated cells were determined by standard assayof resuspended cells. Briefly, 25 μl of urease buffer comprising 0.1 Mcitrate, 2 g/l urea and phenol red 0.01% was added to an equal volume oftreated cell suspension, and the pH of the mixture was determined atroom temperature over a period of 30 mins. In this assay, a change inassay sample colour from yellow to red is indicative of an increase inpH due to breakdown of urea and production of ammonia. Data obtained fortwo independent experiments indicates that UV-C irradiated cells hadresidual urease activity relative to untreated cells, estimated to beless than 10% of the urease activity of untreated H. pylori cells e.g.,prior to UV irradiation.

Consistent with the reduced urease activity of the irradiated cells,SDS/PAGE of extracts from H. pylori cells exposed to UV-C irradiationdemonstrate that the irradiated cells undergo protein degradation, andaggregation of proteins into high molecular weight complexes, comparedto untreated cells (data not shown). In a UV-C dosage range of 1-4J/cm², the level of such degradation and aggregation is dose-dependenti.e., a higher UV-C dose e.g., 2 J/cm² or 4 J/cm², produces increaseddegradation and aggregation (data not shown).

Collectively, the data indicate that UV-C irradiation and optionally,additional freeze-thawing of H. pylori, provides an effective means forinactivating and/or killing H. pylori.

Example 2 Treatment to Inactivate and/or Kill H. pylori Cells—Method II

This example demonstrates the utility of ultraviolet irradiation oroxygen restriction, and optional additional heat treatment followingultraviolet irradiation or oxygen restriction and/or by heat treatmentalone, for inactivating and/or killing H. pylori cells.

Cells of H. pylori strain OND79, or cells of H. pylori strain OND86deposited with the National Measurement Institute (NMI) of Australiaunder Accession No. V14/013016 (described in Example 15), or cells of H.pylori strain J99 were grown on Columbia agar (CBA) plates comprisingColumbia agar base (Product Code CM0311, Thermo Fisher Scientific, OxoidLtd) and 7% (v/v) sterile defibrinated horse blood for 24 hours andharvesting cells by resuspension of grown cells in saline solution [0.9%(w/v) sodium chloride] and then centrifugation, according to standardprocedures. The cells were then resuspended in saline solution, and theconcentration of resuspended cells was adjusted to a measured absorbanceat 600 nm wavelength of 1 optical density (OD) unit per ml. Equalvolumes (1 ml) of resuspended cells were plated onto CBA platescomprising 7% (v/v) sterile defibrinated horse blood. The plates wereincubated at 37° C. in a microaerobic environment containing 5% (v/v)CO₂ and less than 5% (v/v) O₂ for 24 hours if cells were then subjectedUV irradiation or, alternatively, for 18 hours if the cells were thensubjected to oxygen starvation.

After 24 hours at microaerobic conditions plate samples were thensubjected to ultraviolet irradiation using UV-C light, and theirradiated bacteria were then collected from the plates, resuspended inthe saline solution and the concentration of resuspended cells wasadjusted to a measured absorbance at 600 nm wavelength of 20 opticaldensity (OD) unit per ml, as described in Example 1. Optionally, theresuspended cells were then subjected to heat treatment by exposure to afirst elevated temperature of about at 70° C. for 10 minutes immediatelyfollowed by exposure to a second elevated temperature of about 94° C. or95° C. for 5 minutes at normal atmosphere conditions.

Alternatively, after 18 hours incubation in microaerobic conditions asdescribed above, cultured H. pylori cells were then subjected to oxygenrestriction treatment depleting the H. pylori cultures of oxygen bytransferring the plates to plates hermetically sealed jars containinggas sachets (AnaeroGen, AN0025A, ThermoScientific) to generate anaerobicconditions. Plates were then incubated at under anaerobic conditions at37° C. for periods of 24 h or 48 hours or 72 hours. The bacteria whichwere subjected to oxygen starvation treatment were then collected fromthe plates, resuspended in the saline solution and the concentration ofresuspended cells was adjusted to a measured absorbance at 600 nmwavelength of 20 optical density (OD) unit per ml. Optionally, theresuspended cells were then subjected to heat treatment by exposure to afirst elevated temperature of about at 70° C. for 10 minutes immediatelyfollowed by exposure to a second elevated temperature of about 94° C. or95° C. for 5 minutes at normal atmosphere conditions.

Alternatively, H. pylori cells which had been cultured on CBA plates for24 hours at microaerobic conditions as described above were resuspendedin the saline solution and the concentration adjusted to a measuredabsorbance at 600 nm wavelength of 20 optical density (OD) unit per ml.The resuspended cells were then subjected to inactivation and/or killingby heat treatment alone by exposing the cells to a first elevatedtemperature of about 70° C. for 10 minutes and then to a second elevatedtemperature of about 94° C. or 95° C. for 5 minutes at normal atmosphereconditions.

To test for the ability of H. pylori treated cells to replicate,bacterial suspensions of live untreated H. pylori OND86 cells (livecontrol) and bacterial suspensions of H. pylori OND86 cells that weresubjected to ultraviolet irradiation using UV-C light (UV) andoptionally further subjected to heat treatment (UV+heat) or which weresubjected to oxygen starvation treatment for 48 hours (O₂ restriction)and optionally further subjected to heat treatment (O₂restriction+heat), were serially-diluted in saline, plated onto CBAplates, and the plates incubated for 3 days at 37° C. in a microaerobicenvironment containing 5% (v/v) CO₂ and less than 5% (v/v) O₂. Cellcounts were then determined for the various dilutions. Results obtainedfrom two independent experiments are shown in FIG. 2. The resultsindicate that treatment of H. pylori cells by UV irradiation, UVirradiation and heat treatment, oxygen restriction and heat treatmentabolished the ability of treated H. pylori cells to replicate and formcolonies. Although in one independent experiment where H. pylori cellswere subjected to oxygen restriction for 48 hours without further heattreatment resulted in H. pylori colonies on CBA plates, the ability ofthe treated cells to replicate was substantially reduced relative tountreated live H. pylori. In a further independent experiment, H. pyloricells which were subjected to heat treatment alone to inactivate and/orkill the cells consistently generated no colonies on CBA plates (datanot shown), indicating that exposure to heat treatment alone e.g., asdescribed herein also abrogates replication capabilities of H. pyloricells.

To test for the effect of various oxygen restriction treatment periodson the ability of H. pylori cells to replicate, H. pylori OND86 cellswhich were subjected to oxygen restriction for periods of 24 hours, or48 hours or 72 hours without additional heat treatment, were collectedfrom the plates after incubation under anaerobic conditions as describedabove, resuspended in the saline solution and the concentration ofresuspended cells was adjusted to a measured absorbance at 600 nmwavelength of 1 optical density (OD) unit per ml, and seeded onto freshCBA plates after serial 10-fold dilution. Plates were then incubated for3 days at 37° C. in a microaerobic environment containing 5% (v/v) CO₂and less than 5% (v/v) O₂ . H. pylori were able to form colonies after24 hours of oxygen restriction treatment, however, following 48 hours ofoxygen restriction H. pylori cultures demonstrated limited growth on CBAplates, and after 72 hours of oxygen restriction no H. pylori colonieswere formed on the CBA plates (results not shown). These resultsindicate that treatment of live H. pylori cells by oxygen restrictionfor a period of about 48 hour or more e.g., between 48 hours to 72 hoursor more is effective in reducing and/or preventing replication abilityof H. pylori thereby inactivating and/or killing H. pylori cells.

Urease activities of bacterial suspensions of live untreated H. pyloriOND86 cells (live control) and bacterial suspensions of H. pylori OND86cells that were subjected to ultraviolet irradiation using UV-C light(UV) and optionally further subjected to heat treatment (UV+heat) orwhich were subjected to oxygen starvation treatment for 48 hours (O₂restriction) were and optionally further subjected to heat treatment (O₂restriction+heat), were determined by the standard urease test. Briefly,25 μl of urease buffer comprising 0.1 M citrate, 2 g/l urea and phenolred 0.01% was added to an equal volume of live untreated cells andtreated cell suspension, and the pH of the mixture was determinedspectrophotometrically at 560 nm after incubation of the cells at roomtemperature over a period of 5 mins. In this assay, a qualitative ureaseactivity was determined as a measure of metabolic activity of thetreated cells. H. pylori urease enzyme activity was evaluated based on achange in assay sample colour from yellow to red is indicative of anincrease in pH due to breakdown of urea and production of ammonia.

Results of the qualitative urease activity are provided in Table 1below.

TABLE 1 Qualitative Urease Activity. Treatment of H. pylori OND86 cellsUrease activity No treatment ++ (live control cells) UV irradiation −/+(UV) UV irradiation + − heat O₂ restriction ++ O₂ restriction + − heatEnzymatic activity was evaluated based on the change of colour fromyellow to red measured at 560 nm after incubation of the cells at roomtemperature over a period of 5 mins. Four qualitative levels were used;negative, −; weak, −/+; moderate, +, strong ++. Treatment of H. pyloriOND86 cells are as indicated above.

In a second, independent, experiment urease activity of bacterialsuspensions of live untreated H. pylori OND86 cells (live control) andbacterial suspensions of treated H. pylori OND86 cells was performed bythe urease test as above, except that urease enzyme activity wasmeasured at 560 nm after 1 minute incubation of the cells at roomtemperature. The urease activity of the live untreated bacteria was setat 100%, and the relative urease activity reading output for treatedi.e., inactivated and/or killed bacteria was calculated as a percentageof the urease activity measured for the live untreated H. pylori. Inthis experiment suspensions H. pylori OND86 cells were also tested forurease activity following heat treatment alone i.e., by exposure toelevated temperature of about 70° C. for 10 minutes and then about 94°C. or 95° C. for 5 minutes as described above. The results of the ureaseactivity of H. pylori cells subjected to heat treatment alone (Heat),UV-C irradiation (UV) and optionally further heat treatment (UV+heat),oxygen starvation for 48 hours (O₂ res) and optionally further heattreatment (O₂ res+heat), relative to the urease activity readout of thelive untreated cells is shown in FIG. 3.

The results shown in FIG. 2 and the urease activity results shown inTable 1 and FIG. 3 indicate that although treatment to inactivate and/orkill H. pylori e.g., by way of UV treatment alone, UV plus heattreatment, oxygen starvation plus heat, or by heat treatment alone, canabrogate replication ability of H. pylori, the treated cells displayresidual metabolic activity as determined by the urease test.

To further investigate the metabolic activity of treated H. pyloricells, the ability of treated H. pylori cells to respire was evaluatedby measuring the membrane redox potential of treated cells by flowcytometry using the BacLight™ RedoxSensor™ CTC (product catalogue No.B34956) from Invitrogen (Molecular Probes™ Invitrogen detectiontechnologies) according to manufacturer's instructions. Live cells of H.pylori OND86 strain were subjected to heat treatment alone, oxygenstarvation for 48 hours and optionally further heat treatment, or UV-Cirradiation and optionally further heat treatment as described above toinactivate and/or kill the cells. Approximately the equivalent of 10⁷cells per tune were incubated with 5-cyano-2,3-ditolyl tetrazoliumchloride (CTC) in the dark for 6 hours and then fixed by addition of 4%formaldehyde according to manufacturer's instructions. The ratio of theredox potential obtained by FACS analysis for live untreated H. pyloricells relative or cells which were treated by oxygen starvation or UV-Cirradiation relative to the redox potential obtained for the live ortreated cells after heat treatment was calculated to normalize the redoxpotential of the different inactivation and/or killing treatmentregimes. Without being bound by any specific theory of mode of action,the present inventors speculated that heat treatment of H. pylori cellsas described herein may lead to destruction of the majority of metabolicactivity in treated cells and may further result in alteration of cellshape and/or cell aggregation patterns. Accordingly, to take intoaccount variations in FACS cells sorting arising from differences incell shape or cell aggregation that may arise due to heat treatment ofcells, normalization of the redox potential for cells following heattreatment was performed. The results are shown in FIG. 4. The resultsshow that both live and UV-C treated cells were metabolically active andwere respiring before heat treatment (ratio of 0.85 and 1.1,respectively), whereas treatment of live H. pylori cells by exposure tooxygen restriction led to a ratio of 0.1 indicating that treatment ofcells by oxygen starvation significantly attenuated metabolic activityof H. pylori. Treatment by oxygen restriction resulted in 8.5-folddecrease in the redox potential ratio compared to redox potential rationobtained for live cells, but UV-C irradiation had little effect on theredox potential ratio relative to live cells.

Collectively, the herein data indicate that UV-C irradiation andoptionally heat treatment of cells, or oxygen starvation and optionallyheat treatment of cells, provide an effective means for inactivatingand/or killing H. pylori.

Example 3 H. pylori Improves Outcomes of Allergic Asthma in the OVAModel of Allergic Airways Disease

Adult C57BL/6 mice (6 to 8 weeks) were infected with wild-type H. pylori(WT), wild-type H, pylori expressing the asthma inducing antigen (OVA),or treated H. pylori (KD), or left uninfected. The H. pylori inoculacomprised 0.2 ml of a suspension of H. pylori strain OND79 cells insaline solution adjusted to a measured absorbance at 600 nm wavelengthof 20 OD unit per ml. Treated H. pylori were inactivated and/or killedas described in Example 1. Eight (8) weeks later, an allergic asthmaphenotype was induced by sensitized mice with OVA/alum i.p. (day 0 and14) and then challenged with OVA aerosol for 5 days from day 21-25.Control mice were uninfected, sensitised and challenged (positive) oronly sensitised (negative). On day 26 mice received methacholine (MCh)challenge at increasing doses and airway resistance in the lungs wasmeasured. FIG. 5 shows that H. pylori protected mice from induction ofan allergic asthma phenotype.

Example 4 H. pylori Reduces Total Cell Count and Eosinophilia in the OVAModel of Allergic Airways Disease

Adult C57BL/6 mice (6 to 8 weeks) were infected orally by gavage withwild-type H. pylori (WT), wild-type H. pylori-expressing the asthmainducing antigen (HpOVA), treated H. pylori (KD) or left uninfected. TheH. pylori inocula comprised 0.2 ml of a suspension of H. pylori strainOND79 cells in saline solution adjusted to a measured absorbance at 600nm wavelength of 20 OD unit per ml. Treated H. pylori were inactivatedand/or killed as described in Example 1. Eight (8) weeks later, anallergic asthma phenotype was induced by sensitised mice with OVA/alumi.p. (day 0 and 14) and then challenged with OVA aerosol for 5 days fromday 21-25. Control mice were uninfected, sensitised and challenged(positive) or only sensitised (negative). On day 26 mice were sacrificedand bronchioalveolar lung fluid collected. Total cell counts (Panel A)and eosinophil counts (Panel B) in the BALF were enumerated and theaverage number of eosinophils recruited to the lung is represented from10 mice per group. FIG. 6 shows that H. pylori reduces total cell countand eosinophilia.

Example 5 H. pylori Decreases OVA-Specific IgE and OVA-Specific IgGResponse in the OVA Model of Allergic Airways Disease

Adult C57BL/6 mice were infected orally by gavage with wild-type H.pylori (WT), wild-type H. pylori-expressing the asthma inducing antigen(HpOVA), treated H. pylori (KD) or left uninfected. The H. pyloriinocula comprised 0.2 ml of a suspension of H. pylori strain OND79 cellsin saline solution adjusted to a measured absorbance at 600 nmwavelength of 20 OD unit per ml. Treated H. pylori were inactivatedand/or killed as described in Example 1. Eight (8) weeks later, micewere sensitized with 20 μg OVA/1 mg alum i.p. (day 0 and 14) and thenchallenged intranasally with 2 μg OVA in saline for 4 days from day21-24. Control mice were uninfected, sensitised and challenged(positive) or only sensitised (negative). On day 25 mice were bled andOVA-specific IgE (Panel A) and IgG (Panel B) antibodies were measuredfrom serum, diluted 1:60 and 1:6000 respectively, by ELISA. Results areexpressed as the individual and average absorbance at OD₄₀₅ nm. FIG. 7shows that H. pylori decreases OVA-specific IgE (Panel A) andOVA-specific IgG (Panel B) response in the ova model of allergic airwaysdisease.

Example 6 H. pylori Reduces IL-13

Adult C57BL/6 mice were infected orally by gavage with H. pylori (WT),treated bacteria (KD) or left uninfected. The H. pylori inoculacomprised 0.2 ml of a suspension of H. pylori strain OND79 cells insaline solution adjusted to a measured absorbance at 600 nm wavelengthof 20 OD unit per ml. Treated H. pylori were inactivated and/or killedas described in Example 1. As a comparator, H. pylori strain 10700 wasalso tested. 8 weeks later, mice were sensitized with 20 μg OVA/1 mgalum i.p. (day 0 and 14) and then challenged intranasally with 2 μg OVAin saline for 4 days from day 21-24. Control mice were uninfected,sensitised and challenged (positive) or only sensitised (negative). Onday 25 bronchioalveolar lung fluid (BALF) was collected from the lungsof anaesthetised mice. IL-13 was measured from undiluted BALF usingcytokine bead array and expressed as the average of 10 mice per group inpg/ml. FIG. 8 shows that IL-13 is reduced in the lungs of H.pylori-infected mice in the allergic asthma model.

Example 7 H. pylori Reduces OVA-Specific CD8 T Cells

Adult C57BL/6 mice were infected orally by gavage with ˜1×10⁹ CFU H.pylori (WT) or left uninfected for 8 months then received 2 doses of 20μg OVA/2 mg alum on day 0 and 28. The H. pylori inocula comprised 0.2 mlof a suspension of H. pylori strain OND79 cells in saline solutionadjusted to a measured absorbance at 600 nm wavelength of 20 OD unit perml. One day prior to OVA/alum challenge, mice received 5×10⁴ MACSpurified CD8 OT-I cells i.v. Spleens were harvested on day 35 and singlecell suspension of spleen cells was stimulated with SIINFEKL peptide for4 hours in presence of BrefA. Intracellular cytokine staining wasperformed to measure IFNγ secretion by FACS. CD8 OT-I cells wereidentified by CD45.1 expression. Colonization results from the stomachshowed that all WT infected mice were colonized. H. pylori reduces theOVA-specific CD8 T cell response and impairs function of OVA-specificCD8 T cells. FIG. 9 shows the decreased number (panel A) and function(panel B) of OVA-specific CD8 T cells in H. pylori infected micecompared to control mice after OVA/alum challenge.

Example 8 H. pylori Decreases Antigen-Specific IgG

Adult C57BL/6 mice were infected orally by gavage with ˜1×10⁹ CFU H.pylori (WT) or left uninfected for 8 weeks then injected i.p. with 20 μgOVA/alum. 14 days later serum was collected and OVA-specific IgGdetermined by ELISA.

In some mice a secondary i.p. dose of OVA/alum was administered at day14. No differences in OVA-specific IgG titres was observed at day 21 (7days after boost). Mice are able to overcome H. pylori-mediated immunesuppression in the presence of sufficient immunological stimulus. FIG.10 (panel A) shows decreased antigen-specific IgG in H. pylori-infectedcompared to control mice after primary OVA/alum challenge. FIG. 10(panel B) shows antigen specific IgG response 7 days after secondarychallenge.

Example 9 H. pylori Reduces Responsiveness of CD4 and CD8 T Cells

Adult C57BL/6 mice were infected orally by gavage with ˜1×10⁹ CFU H.pylori (WT) or left uninfected. 7 months after challenge spleen cellswere isolated and single suspensions of cells stimulated withPMA/ionomycin for 4 hours in the presence of Brefeldin A. Numbers ofIFNγ CD4+ and CD8+ T cells were assessed using intracellular cytokinestaining and FACS. FIG. 11 shows the reduced responsiveness of CD4 andCD8 T cells from H. pylori infected mice to non-specific stimulus.

Example 10 Effect of H. pylori Colonisation in the Neonatal AllergicAsthma Model

5-day old female C57BL/6 mice (n=5-10) were fed ˜10⁹ CFU live H. pylorifor 5 consecutive days or left uninfected. 8 weeks later, mice weresensitized with 2 doses of 50 μg OVA/1 mg alum i.p. (day 0 and 14) andthen challenged with OVA aerosol for 5 days from day 21-25. Control micewere uninfected, sensitised and challenged (positive control, i.e.,untreated allergic mice) or only sensitised (negative control, i.e.,untreated healthy mice). On day 26 mice received metacholine (MCh) atincreasing doses and airway hyperresponsiveness (AHR) of lung tissue wasmeasured and bronchio-alveolar lung fluid (BALF) collected.

FIG. 12, in panel A, shows the results in which AHR results areexpressed as the average cmH2o.s/ml per group of mice. Statisticalsignificance was determined using a one-sided student's t-test assuminga normal Gaussian distribution where p<0.5. The non-parametric Wilcoxonrank test that is suitable for non Gaussian distribution showedstatistical significance at the three highest concentration of MCh. InFIG. 12, panel B, total cell infiltrate from the lungs was determine andis expressed at the average number of live total cells from BALF pergroup of mice. Bars represent standard deviation from the mean.Statistical significance was determined using a one-sided student'st-test assuming a normal Gaussian distribution where p<0.5.

The results herein demonstrate that H. pylori reduces symptoms ofallergic asthma. FIG. 12 (panel A) shows that airway resistanceincreased in allergic adult mice not infected with live H. pylori,whereas mice challenged with live H. pylori from day 5 exhibitedcomparable airway resistance to that of non-allergic mice. FIG. 12(panel B) further demonstrates that H. pylori colonization preventscellular infiltration in the lungs after allergen challenge and that thetotal cell count was similar to non-allergic control mice. Accordingly,live H. pylori protects neonatal mice from developing allergic asthma inresponse to allergen exposure later in life and reduces cellularinfiltrate in the lungs.

FIG. 12 demonstrates that H. pylori colonization e.g., in neonatesimproves outcomes of allergic airways disease and reduces risk ofdeveloping allergic airway disease, for example as shown using theneonatal allergic asthma model described herein.

Example 11 Effect of H. pylori on Immunological Outcome in a NeonatalAllergic Asthma Model

The present inventors inter alia conduct a side-by-side comparison ofthe effects on protection against allergic disease, such as allergicairway disease, achieved by administration of live colonizing bacteriaor repeated oral administration of treated H. pylori to neonatal mice.The H. pylori inocula comprised 0.2 ml of a suspension of H. pyloristrain OND79 cells in saline solution adjusted to a measured absorbanceat 600 nm wavelength of 20 OD unit per ml. Treated H. pylori wereinactivated and/or killed as described in Example 1. Briefly, 5-day oldC57BL/6 mice (n=10) were fed ˜10⁹ CFU live H. pylori for 5 consecutivedays or treated bacteria (for 3 days per week, for 10 weeks) or leftuntreated. 8 weeks later, mice were sensitized with 2 doses of 50 μgOVA/1 mg alum i.p. (day 0 and 14) and then challenged with OVA aerosolfor 5 days from day 21-25. Control mice were uninfected, sensitised andchallenged (positive control i.e., untreated allergic mice) or onlysensitised (negative control i.e., untreated healthy mice). On day 26mice were sacrificed and serum and bronchio-alveolar lung fluid (BALF)collected. Total cell infiltrate per group of mice in the lungs wasdetermined and is expressed as the individual and average number of livetotal cells from BALF. As shown in FIG. 13 administration of eithertreated or live H. pylori effectively reduced cellular infiltration inthe lungs of H. pylori treated mice (Panel A). In addition, allergen(OVA)-specific IgE antibodies were measured by standard ELISA methodsfrom serum diluted 1:60. Antibody titres were expressed as theindividual and average absorbance at OD₄₀₅ nm. As shown in FIG. 13(panel B), administration of either treated H. pylori or live H. pylorialso reduced allergic allergen-specific IgE antibodies in H. pyloritreated subjects. *Statistical significance was determined using aone-sided student's t-test assuming a normal Gaussian distribution wherep<0.5. Collectively, the data illustrated in panels (A) and (B) of FIG.13 demonstrate that administration of treated i.e., inactivated and/orkilled (or live) H. pylori is effective in reducing allergicinflammation and/or allergic immune responses.

Inflammatory cytokines, IL-5 and IL-13 were measured from undiluted BALFusing a cytokine bead array kit and results are expressed as the averageconcentration of cytokine per group in pg/ml and shown in FIG. 13(panels C and D). The results shown in FIG. 13 (panels C and D)demonstrate that administration of treated (or live) H. pylori wassuccessful in reducing production of cytokine mediators and biologicalmarkers of asthma and allergic respiratory disease, IL-5 and IL-13, inthe lungs.

In another experiment as shown in FIG. 14, the present inventors alsodemonstrated that adult and neonatal mice administered with treated(i.e., inactivated and/or killed) or live H. pylori had reduced allergicairway resistance response. Lung airway hyperresponsivness (AHR) wasmeasured using 5-day old female C57BL/6 mice (n=5-10) as well as adultC57BL/6 mice (6-8 weeks, n=10) essentially as described in Example 10.In brief, neonatal and adult mice were fed ˜10⁹ CFU/dose of treatedbacteria, 3 times per week for 8 weeks or fed ˜10⁹ CFU/dose of livefreshly cultured bacteria for 6 consecutive days. Treated H. pylori wereinactivated and/or killed as described in Example 1. On day 0 and 14,all mice received intraperitoneal OVA/alum. The allergic asthmaphenotype was induced with 1% OVA aerosol for 5 consecutive days fromday 21. Control mice were uninfected, sensitised and challenged(positive control, i.e., untreated allergic mice) or only sensitised(negative control, i.e., untreated healthy mice). In other words,positive and negative controls did not receive bacteria. On day 26 micereceived metacholine (MCh) at increasing doses and airwayhyperresponsiveness (AHR) of lung tissue in response to MCh challengewas measured, and mice were sacrificed.

As shown in FIG. 14, panel A, allergic adult mice which did not receiveH. pylori (positive control) showed elevated airway resistance afterallergen challenge compared with airway resistance of adult mice whichreceived a formulation of treated H. pylori or live H. pylori. As shownin FIG. 14, panels B and C, allergic neonatal mice which did not receiveH. pylori (positive control) showed elevated airway resistance afterallergen challenge compared with airway resistance of neonatal micewhich received a formulation of treated H. pylori or live H. pylori. Theresults shown in panels A, B and C of FIG. 14 represent threeindependent experiments, and demonstrate that H. pylori can reduce orattenuate allergic response e.g., of allergic airway disease such asasthma in response to allergen in both adults and neonatal subjects.These results further demonstrate that this effect occurs equally aswell when either live H. pylori or inactivated and/or killed H pyloriare used. In other words, the results demonstrate that inactivatedand/or killed H. pylori bacteria are as effective as live H. pyloribacteria in reducing or attenuate allergic response e.g., of allergicairway disease such as asthma in response to allergen in both adults andneonatal subjects, thereby protecting subjects from allergic diseasesuch as allergic as asthma.

Example 12 Inactivated and/or Killed H. pylori Cells do not have theSame Colonization Capability of Live H. pylori Cells

This example demonstrates the utility of treatment to inactivate and/orkill H. pylori cells in reducing the efficacy of H. pylori cells incolonizing the gastric mucosa of allergic subjects in adult allergicasthma model.

Adult C57BL/6 mice (6 to 8 weeks, n=10) were infected orally by gavagewith ˜1×10⁹ CFU of OND79 H. pylori (WT) or treated H. pylori three (3)times per week for a duration of eight (8) weeks. The H. pylori inoculacomprised 0.2 ml of a suspension of H. pylori strain OND79 cells insaline solution adjusted to a measured absorbance at 600 nm wavelengthof 20 OD unit per ml. Treated H. pylori were inactivated and/or killedas described in Example 1. At the end of the 8 weeks period, mice weresensitized with 2 doses of 50 μg OVA/1 mg alum i.p. (day 0 and 14) andthen challenged with OVA aerosol for 5 days from day 31-35. Control micewere uninfected, sensitised and challenged (positive) or only sensitised(negative). Mice were sacrificed on day 36 and stomach tissue harvested.Stomachs were dissected along the greater curvature and residual foodremoved by gently washing with PBS. Opened stomachs were placed in 500μl PBS and homogenized with a 5 mm stainless steel bead for 30 secondsat a frequency of 30 (Qiagen TissueLyser II). Samples were furtherhomogenized for 2 min at a frequency of 10. Serial dilutions ofhomogenates were plated on BHI agar plates supplemented withamphotericin B (8 μg/ml), trimethoprim (5 μg/ml) and vancomycin (6μg/ml), nalidixic acid (10 μg/ml), polymyxin B (10 μg/ml) and bacitracin(200 μg/ml). Plates were placed in gas-controlled chambers containingtwo Campygen kit gas packs (Product Code CN0025A, Thermo FisherScientific, Oxoid Ltd) and incubated at 37° C. Bacterial growth wasdetermined 5-7 days post plating. Results of H. pylori colonization ofthe gastric mucosa in infected mice are shown in FIG. 15 and areexpressed as the number of colony forming units (CFU) per stomach permouse.

The results in FIG. 15 demonstrate that although live untreated H.pylori were able to colonize gastric mucosa of allergic mice, treated H.pylori did not colonize gastric mucosa of infected allergic adult mice.This demonstrates that treated H. pylori cells do not have the samecolonization capability as a live bacterium having the same genotype.

The inventors also tested the effect of H. pylori colonisation in theneonatal allergic asthma model. In particular, the inventors haverepeated the above experiment with the exception that instead of usingadult mice, 5-day old female C57BL/6 mice (n=5-10) were infected orallyby gavage with ˜1×10⁹ CFU of OND79 H. pylori (WT) or treated H. pylorithree (3) times per week for a duration of eight (8) weeks. The H.pylori inocula comprised 0.2 ml of a suspension of H. pylori strainOND79 cells in saline solution adjusted to a measured absorbance at 600nm wavelength of 20 OD unit per ml. Treated H. pylori were inactivatedand/or killed as described in Example 1. At the end of the 8 weeksperiod mice were treated as above. The results obtained show thatcolonization with live untreated (WT) H. pylori was achieved in 1 out of5 neonatal mice at the commencement of the study. On the other hand, nocolonization was observed for any mice infected with treated H. pylorias demonstrated by lack of any detectable H. pylor CFU on BHI agarplates plated with undiluted and serial dilutions of 1:10 and 1:100 ofhomogenised stomach samples (data not shown). These results confirm thattreated H. pylori cells which are inactivated and/or killed also havereduced colonization capability relative to a live H. pylori having thesame genotype in neonatal subjects.

Example 13 Inactivated and/or Killed H. pylori Cells are Unable toColonize the Gastric Mucosa

This example supports the findings in Example 12 and furtherdemonstrates that treatment to inactivate and/or kill H. pylori cellsabrogates ability of H. pylori cells in colonizing the gastric mucosa ofadult mice.

Adult C57BL/6 mice (6 to 8 weeks, n=5) were repeatedly inoculated orallyby gavage with approximately 1×10⁹ CFU of treated OND79 H. pylori 3times per week for 2 weeks. The H. pylori inocula comprised 0.2 ml of asuspension of treated H. pylori strain OND79 cells in saline solutionadjusted to a measured absorbance at 600 nm wavelength of 20 OD unit perml. Treated H. pylori were inactivated and/or killed by subjecting liveH. pylori cells to ultraviolet irradiation using UV-C light andoptionally further subjected to heat treatment, or by subjecting live H.pylori cells to oxygen starvation treatment for 48 hours and optionallyfurther subjected to heat treatment, as described in Example 2.

To determine the level of colonization, stomach tissue was harvestedfrom animals 2 weeks after final oral inoculation. Stomachs weredissected along the greater curvature and residual food removed bygently washing with PBS. Opened stomachs were placed in 500 μl PBS andhomogenized with a 5 mm stainless steel bead for 30 seconds at afrequency of 30 (Qiagen TissueLyser II). Samples were furtherhomogenized for 2 min at a frequency of 10. Serial dilutions ofhomogenates were plated on H. pylori selective (DENT's supplement,nalidixic acid and bacitracin) F12 agar medium plates. Plates wereincubated as described above and after three days of incubation at 37°C. (Anoxomat, 83% N₂, 7% CO₂, 6% O₂ and 4% H₂) and single colonies werecounted to determine bacterial growth 5-7 days post plating.

Efficacy of infection and colonization of the mouse gastric mucosa fortreated H. pylori was assessed based on the number of colony formingunits (CFU) per stomach. The results are shown in FIG. 16 anddemonstrate that treatment of H. pylori by UV-C irradiation andoptionally heat treatment, or by oxygen starvation for 48 hours andoptionally further heat treatment abolishes the colonization capabilityof H. pylori. These results confirm the findings in Example 12 andfurther demonstrate that it is possible to inactivate and/or kill H.pylori and prevent colonization by H. pylori by more than merely onemeans of treating live H. pylori cells.

Example 14 Immunological Efficacy of Inactivated and/or Killed H. pyloriin Neonatal Allergic is not Strain Specific

This example demonstrates a side-by-side comparison of the effects onimmunological protection against allergic disease, achieved byadministration of treated i.e., inactivated and/or killed H. pyloristrains from different geographical origins and belonging to geneticallyremoved ancestral populations of H. pylori. There are identified 6distinct ancestral populations of H. pylori identified by multi-locussequence typing analysis and have been named ancestral European 1,ancestral European 2, ancestral East Asia, ancestral Africa1, ancestralAfrica2, and ancestral Sahul. H. pylori strain OND79 used in thisexample is a European strain, and H. pylori strain J99 used in thisexample is an African strain.

Live H. pylori OND79 cells or H. pylori J99 cells were inactivatedand/or killed by UV-C irradiation treatment as described Examples 1 and2. Treated H. pylori OND79 cells and treated H. pylori J99 cells wereadministered to 5-day old C57BL/6 mice, and mice were sensitized andchallenged with allergen (OVA) by following the same method described inExample 11. Control mice were uninfected, sensitised and challenged(positive control i.e., untreated allergic mice) or only sensitised(negative control i.e., untreated healthy mice). On day 26 mice weresacrificed and serum and collected. In addition, allergen (OVA)-specificIgE and IgG antibodies were measured by standard ELISA methods fromserum diluted 1:60. Antibody titres were expressed as the individual andaverage absorbance at OD405 nm.

As shown in FIG. 17 administration of either UV-C treated H. pyloriOND79 cells or UV-C treated H. pylori J99 cells reduced allergicallergen-specific IgE and IgG antibodies in mice. These resultsdemonstrate that efficacy conferred by administering treated i.e.,inactivated and/or killed H. pylori (such as UV-C treated H. pylori) inallergic asthma mouse model is not strain specific. This is becausetreated i.e., inactivated and/or killed H. pylori of different originswere effective in reducing allergic immune responses to allergenrelative to untreated allergic mice.

Example 15 Production of a H. pylori Strain Passaged in a Human Host forUse in the Compositions and/or Methods of the Invention

This example demonstrates the production and characterization of apassaged strain or derivative of H. pylori strain OND79 obtained afterpassaging in a human host. The resulting passaged or derivative strainof H. pylori is suitable for treatment to inactivate and/or kill thecells and use in the compositions and/or methods of the invention.

Expansion of H. pylori OND79 for Administration to Human

H. pylori OND79 strain was expanded for human administration by thefollowing method. Specifically, commercially available PyloriAgar (PA)plates (from BioMerieux, France) were purchased for culture of H. pyloriOND79 strain to prepare an inoculum of the OND79 strain for humanchallenge. To this effect, a glycerol stock vial of H. pylori OND79strain (Heart Infusion [HI] broth containing 20% (v/v) glycerol and 10%(v/v) of H. pylori OND79 cells) which had been stored at −80° C., wasthawed and inoculated onto 5 PA agar plates. The bacteria-inoculatedplates were subjected to an atmosphere evacuation/replacement cycleusing an Anoxomat (ANCTS2, Mart Microbiology, Drachten, The Netherlands)to generate micro-aerobic conditions (approximately 83% N₂, 7% CO₂, 6%O₂ and 4% H₂) and were incubated at 37° C. for 72 h. The total platecontent was then expanded onto new PA plates. Bacteria were harvestedand suspended in 1 ml of sterile saline solution (0.9%). Six plates werethen inoculated with 100 μl of the bacterial suspension. Cells wereevenly distributed on the plates with a sterile disposable loop andincubated under micro-aerobic conditions at 37° C. for 72 h as describedabove. After 24 h four plates were harvested into 10 ml of regular beefstock solution (1 gram [Continental, Unilever, Australia] in 80 mlpreheated water that was filter-sterilized through a 0.2 μm Milliporesyringe filter). Biochemical tests including urease, catalase andoxidase tests as well as Gram staining were performed to confirm thatthe stock solution comprised a pure H. pylori culture. The bacterialstock solution was placed on ice and transported to the Department ofGastroenterology and Hepatology at Sir Charles Gairdner Hospital (SCGH)(Western Australia) for administration to the human subject volunteerunder SCGH Human Research Ethics Committee approval #2009-062.Approximately 10⁹ viable bacteria were then administered orally to ahuman subject volunteer by ingestion. Two weeks post administration thepatient underwent endoscopy and a gastric biopsy taken to confirm H.pylori colonization of the gastric mucosa and the patient was leftuntreated for a period of at least 12 weeks post administration tomaintain H. pylori gastric colonization in the human subject.

Isolation of H. pylori OND86 Strain from Human Gastric Biopsies

Twelve weeks post bacterial inoculation the human subject underwent anendoscopy to collect several gastric biopsies. One gastric antrum biopsyobtained from the subject was processed by homogenization [Qiagen TissueLyser] and serially diluted in sterile physiological saline forculturing bacteria from the gastric biopsy on H. pylori selective(DENT's supplement, nalidixic acid and bacitracin) F12 agar mediumplates (Thermoscientific, Australia). Bacterial cultures were incubatedunder micro-aerobic conditions (approximately 83% N₂, 7% CO₂, 6% O₂ and4% H₂) at 37° C. for 72 h as described above, and then single bacterialcolonies were isolated and expanded three to four times to produceclonal cultures of a H. pylori strain isolated from the gastric biopsyof the patient. Pure clonal H. pylori cultures were verified by Gramstaining and biochemical tests as above and the expanded single colonieswere frozen in triplicates and stored at −80° C. in F12 broth with 20%(v/v) vegetable glycerol (freezing medium). A pure clonal culture of H.pylori strain derived from H. pylori OND79 after passage in the humansubject was named H. pylori OND86 strain and a sample was deposited on10 Jun. 2014 with the National Measurement Institute (NMI), 1/153Bertrie Street, Port Melbourne, Victoria, Australia, pursuant to theprovisions of the Budapest Treaty, and allocated the NMI Accession No.V14/013016.

Characterization of Clinical Isolates of H. pylori Derived from OND79Following Passage in a Human Host

Analysis of genomic DNA diversity among the H. pylori parent strainOND79 and six clinical isolates of H. pylori obtained as described abovefrom gastric biopsies of three human volunteers administered with theparent OND79 strain, was performed using a PCR-based Randomly AmplifiedPolymorphic DNA (RAPD) fingerprinting method as described by Akopyanz etal., (1992) Nucleic Acids Research, 20:5137-5142. The six clinicalisolates of H. pylori were labelled “#1157 clone 1”, “#1157 clone 9”,“#86198 clone 1”, “#86198 clone 9”, “#45156 clone 1” and “#45156 clone9”. Clinical isolates #1157 clone 1, and #1157 clone 9 represent twoclonal isolates obtained from the same gastric biopsy of the same humansubject (volunteer 1) administered with the parent OND79 strain.Similarly, clinical isolates #86198 clone 1, and #86198 clone 9represent two clonal isolates obtained from the same gastric biopsy ofthe same human subject (volunteer 2) administered with the parent OND79strain. Clinical isolates #86198 clone 1, and #86198 clone 9 representtwo clonal isolates obtained from the same gastric biopsy of the samehuman subject (volunteer 3) administered with the parent OND79 strain. Apure clonal culture of H. pylori clinical isolate #1157 clone 9 waschosen for deposit as H. pylori OND86 strain under NMI Accession No.V14/013016 described above.

RAPD fingerprinting was performed on the H. pylori parent strain OND79and the six clinical isolates using either the primer “1254” set forthin SEQ ID NO: 3 and having the sequence 5′-CCG CAG CCA A-3′, or theprimer “1281” set forth in SEQ ID NO: 4 and having the sequence 5′-AACGCG CAA C-3′. As shown in FIG. 18, genomic RAPD fingerprinting wasidentical for the parent OND79 strain and for each clinical isolate ofthe human passage derivative strain including the deposited OND86strain. Such result indicates that a H. pylori strain that has beenpassaged through an animal host, such as a human host passaged clinicalisolates have similar, if not identical, genetic makeup as the parentOND79 strain.

Example 16 H. pylori Strain Derived from OND79 that has been Passaged ina Human Shows Strong Colonization Efficacy of the Gastric Mucosa inInfected Animals

This example demonstrates that a passaged strain or derivative of H.pylori strain OND79 obtained after passaging in a human host is able tocolonize the gastric mucosa of animals.

Adult C57BL/6 mice (n=5) were orogastrically inoculated withapproximately 1×10⁹ live bacteria from pure cultures of each one of thesix clinical isolates of the H. pylori obtained after passaging H.pylori OND79 in a human host described in Example 15 i.e., #1157 clone1, #1157 clone 9, #86198 clone 1, #86198 clone 9, #45156 clone 1 and#45156 clone 9. To determine the level of colonization of the 6 clinicalisolated (including a clinical isolated of the H. pylori OND86 straindeposited under NMI Accession No. V14/013016) stomach tissue washarvested from animals 2 weeks after bacterial administration. Stomachswere dissected along the greater curvature and residual food removed bygently washing with PBS. Opened stomachs were placed in 500 μl PBS andhomogenized with a 5 mm stainless steel bead for 30 seconds at afrequency of 30 (Qiagen TissueLyser II). Samples were furtherhomogenized for 2 min at a frequency of 10. Serial dilutions ofhomogenates were plated on H. pylori selective (DENT's supplement,nalidixic acid and bacitracin) F12 agar medium plates. Plates wereincubated under micro-aerobic conditions (Anoxomat, approximately 83%N2, 7% CO2, 6% O2 and 4% H2) at 37° C. for 72 h as described above, andthen single colonies were counted (i.e., bacterial growth) wasdetermined 5-7 days post plating. Efficacy of infection and colonizationof the mouse gastric mucosa for each one of the six isolates of the H.pylori derivative strain obtained after passaging H. pylori OND79 in ahuman host was measured based on the number of colony forming units(CFU) per stomach. As shown in FIG. 19, all six clinical isolates(including the deposited H. pylori OND86 strain) were able toeffectively infect and colonize the mouse gastric mucosa.

Example 17 H. pylori Strain Derived from OND79 that has been Passaged ina Human Host Such Demonstrate Strong Efficacy in Colonizing the GastricMucosa of Animals

This example demonstrates that a passaged strain or derivative of H.pylori strain OND79 obtained after passaging in a human host inducesspecific anti-H. pylori IgG antibody in animals.

The adult C57BL/6 mice which were orogastrically inoculated with thelive bacteria from pure cultures of each one of the six clinicalisolates of the H. pylori described in Example 14 were also used todetermine the immunogenicity efficacy of the six clinical isolates ofthe H. pylori described above. Serum was collected from the mice at theend point of the colonization experiment described in Example 16.Ninety-six well plates (Nunc Maxisorb) were coated with 10 μg/ml H.pylori X47 strain cell lysate and incubated overnight at 4° C. Plateswere then washed 5 times in PBS/0.05% Tween-20 and blocked with 2%bovine serum albumin (BSA) for 2 hours at 37° C. Plates were washedtwice and serum samples (1/20 dilution) were added to the wells induplicate. The plates were then incubated for 1 h at room temperature(RT), subsequently washed and detection antibody (anti-mouse IgGconjugated to alkaline phosphatase, 1/1000, Sigma) was added. Plateswere further incubated for 1 h at RT then washed. Plates were developedusing p-NPP for 60 min before the reaction was stopped with 2M NaOH.Antibody titres were expressed as the OD value measured at 405 nm. Asshown in FIG. 20, all six clinical isolates (including the deposited H.pylori OND86 strain) were able to induce antibody specific immuneresponses to H. pylori.

Taken together the results presented herein demonstrate inter alia thatadministration of live, killed or inactivated forms of H. pylori to amammalian subject can modulate the mammalian host immune responses tosupress or attenuate allergic immune responses to an allergen, and/orsuppress or attenuate allergic airway disease such as allergic asthma.The results presented herein also inter alia demonstrate thatformulations comprising live, killed, or inactivated H. pylori canprevent development of an allergic immune response or allergic diseasesuch as allergic airway disease, and can have utility as animmunotherapy in children such as neonates and/or juveniles to preventor limit the atopic march and the progression of allergic disease in asubject e.g., prevent or limit progression of allergic disease inchildren with eczema to food allergy and/or severe asthma later in life.Furthermore, as demonstrated herein efficacy in supressing or attenuateallergic immune responses to an allergen conferred by killed and/orinactivated H pylori is not strain specific.

Further Non-Limiting Examples of the Invention

A composition comprising an isolated H. pylori cell, a cell lysatethereof or combination thereof, optionally further processed to producea processed H. pylori preparation such as an extract prepared from wholeH. pylori cells or proteins isolated from H. pylori cells which arepartially or completely purified and/or pre-treated, and apharmaceutically accepted carrier, wherein said H. pylori cell is eitherkilled or incapable of colonizing the mucosa of said mammal. The term“composition” as used herein refers a therapeutically-effective orprophylactically-effective amount of the H. pylori bacteria or H. pyloricell lysate or combination thereof which is optionally in admixture witha pharmaceutically acceptable carrier, excipient or diluent suitable forwhich are administered to a mammal. Generally, the composition isprepared to be administered as a therapeutically effective amount. Apharmaceutically acceptable carrier are any organic or inorganic inertmaterial suitable for administration to a mammalian musoca, e.g., water,gelatin, gum arabic, lactose, starch, magnesium stearate, talc,vegetable oils, polyalkylene-glycols, petroleum jelly and the like,optionally further comprising one or more other pharmaceutically activeagents, flavouring agents, preservatives, stabilizers, emulsifyingagents, buffers and the like, added in accordance with acceptedpractices of pharmaceutical compounding. A “therapeutically effectiveamount” of the composition of the present invention is understood tocomprise an amount effective to elicit the desired response e.g.,anergy, but insufficient to cause a toxic reaction. As used herein, theterm “anergy” refers to either a diminished immune reaction, or theabsence of an immune reaction to an antigen as revealed by the lack ofan appropriate immune response, possibly entailing a reversibleanti-proliferative state which results in decreased responsiveness of animmune cell or cells to an antigen. The term “cell lysate thereof” asused herein refers to a preparation of the H. pylori cells of thepresent invention, in which the H. pylori cells have been disrupted suchthat the cellular components of the bacteria are disaggregated orliberated. Persons skilled in the art would be well aware of techniquesfor producing bacterial cell lysates. For example, H. pylori cells arepelleted and then resuspended in, for example, Dulbecco's phosphatebuffered saline (PBS; 10 mM phosphate, 0.14 M NaCl, pH 7.4) andsubjected to sonication on ice with a W-375 sonication Ultrasonicprocessor (Heat Systems-Ultrasonics, Inc., Farmingdale, N.Y.) at 50%duty cycle with pulse and strength setting 5 for three 1 min sessions.If required, insoluble material and unbroken bacterial cells can then beremoved by centrifugation. Alternatively, H pylori cells are collectedby centrifugation and resuspended in PBS and then lysed by passagethrough a French press (SLM Instrument Inc., Urbana, Ill.) at 20,000LB/in. Again, if required, the bacterial lysate are centrifuged at102,000×g for 10 minutes to remove bacterial debris and/or filteredthrough a 0.45 μM membrane (Nalgene, Rochester, N.Y.). Another method ofproducing cell lysate of H. pylori involves freezing and thawing ofbacterial pellets in the presence of lysozyme. A particular example of aH. pylori cell lysate is the soluble fraction of a sonicated culture ofthe H. pylori, e.g., obtained after filtration. Alternatively or inaddition, H. pylori are fragmented using a high-pressure homogenizer(e.g. Avestin model EmulsiFlexC5). Optionally, the cell lysate isfurther inactivated by treatment with formalin, or a comparable agent.Alternatively, the immunotherapy composition according to the presentinvention is obtained by fractionation and/or purification of one ormore proteins from a lysate of H. pylori culture medium. Obviously, aperson skilled in the art will appreciate that if a cell lysate is to beused in the inventive methods described herein there is no need toinactivate or “kill” the H. pylori as it will already be disrupted;however, as described supra or infra, the whole H. pylori it needs to beeither killed or incapable of colonizing the mucosa of said mammal.

A composition consisting essentially of an isolated H. pylori cell and acell lysate thereof together with a pharmaceutically accepted carrier.The terms “composition” and “cell lysate” have the meaning given inparagraph 1.

A composition for use in preventing or treating allergy in a mammalcomprising an isolated H. pylori cell, a cell lysate thereof orcombination thereof and a pharmaceutically accepted carrier, whereinsaid H. pylori cell is either killed or incapable of colonizing themucosa of said mammal. The terms “composition” and “cell lysate” havethe meaning given in paragraph 1 supra. A killed H. pylori is in a stateof irreversible bacteriostasis. While the H. pylori cell retains itsstructure and thus retains, for example, the immunogenicity,antigenicity, and/or receptor-ligand interactions associated with awild-type H. pylori cell, it is not capable of replicating. There arevarious methods known in the art to produce killed (whole) bacteriaincluding H. pylori, such as exposure to ultraviolet (UV) irradiation,exposure to extreme heat and/or pressure and/or infection with abacteriophage. In some embodiments, the killed or inactivated H. pylorimay remain metabolically active e.g., it may wholly retain or partiallyretain a cell wall and a cell membrane and certain enzymatic functionssuch as the presence of catalase and superoxide dismutase (SOD)activities for free radical harvesting, however be incapable ofcolonizing the gastric mucosa of a subject to whom it is administered. Apreferred method of producing killed or inactived H. pylori is by heat,UV irradiation, pressure or chemical means. Exemplary means ofinactivation by irradiation include exposure to ultraviolet irradiationor gamma irradiation. Once a killed or inactivated H. pylori strain, orH. pylori strain that is naturally incapable of colonizing the mucosa ofa mammal, or H. pylori cell lysate has been produced, it are formulatedin to a composition of the present invention.

A composition according to any one of paragraphs 1 to 3, wherein the H.pylori is killed before use in the invention by, for example,inactivating or killing the strain, or wherein the strain is naturallyincapable of colonizing the mucosa of a mammal.

A composition according to any one of paragraphs 1 to 4, wherein the H.pylori is a cagA-deficient or cagA⁻ strain, and preferably a strain thatis also positive for toxigenic s1 and m1 alleles of the VacA gene. Theterms “cagA⁻,” “cagA minus,” “cagA deficient” and the like refer to theabsence of the H. pylori virulence factor cagA (cytotoxin-associatedgene A), which is a 120-145 kDa protein encoded on the 40 kb cagpathogenicity island (PAI) (Hatakeyama & Higashi, (2005), CancerScience., 96: 835-843). H. pylori strains are divided into cagA⁺(positive) or cagA⁻ (negative) strains, of which around 60% of H. pyloriisolates in Western countries are positive, whereas the majority of EastAsian isolates are negative e.g., Hatakeyama & Higashi, (2005).

A composition according to any one of paragraphs 1 to 5, wherein the H.pylori has the characteristics of a strain of H. pylori selected fromthe group consisting of OND737, as deposited in the National MeasurementInstitute under Accession No. V09/009101; OND738, as deposited in theNational Measurement Institute under Accession No. V09/009102; OND739,as deposited in the National Measurement Institute under Accession No.V09/009103; OND248, as deposited in the National Measurement Instituteunder Accession No. V10/014059; OND256 as deposited in the NationalMeasurement Institute under Accession No. V10/014060, OND740 asdeposited in the National Measurement Institute under Accession No.V09/009104; OND79 as deposited in the National Measurement Instituteunder Accession No. V13/023374 and/or OND86 as deposited in the NationalMeasurement Institute under Accession No. V14/013016, or passagedstrain, a mutant or a derivative thereof. The term “mutant” or“derivative” as used herein, refers to H. pylori which is produced fromor derived from a strain of H. pylori described herein and as such hasgenomic DNA at least about 80%, preferably at least about 90%, and mostpreferably at least about 95%, identical to that of H. pylori strainOND737, OND738, OND739, OND740, OND248, OND256, OND79 or OND86.

A composition according to any one of paragraphs 1 to 6, wherein the H.pylori has been passaged through an animal host before it is inactivatedfor use in the present invention.

A composition according to any one of paragraphs 1 to 7, wherein the H.pylori is further genetically modified prior to being inactivated tocomprise one or more nucleic acid molecule(s) encoding at least oneheterologous antigen or a functional fragment thereof. This means thatthe H. pylori will generally express the antigen before it isinactivated. A “genetically modified” H. pylori refers to a H. pyloribacterium that differs in its phenotype and/or genotype from that of thecorresponding wild type H. pylori in that it comprises an alteration toor an addition to the genetic makeup present in H pylori. Methods forthe genetic modification of the H pylori are well-known in the art: See,for example, Sambrook & Russell, (2001), “Molecular Cloning—A LaboratoryManual”, Cold Spring Harbor Laboratory Press, New York, 3^(rd) Edition.An “isolated genetically modified H. pylori cell may be present in amixed population of H. pylori cells. In some embodiments, thegenetically modified H pylori will comprise one or more nucleic acidmolecule(s) encoding at least one heterologous antigen or a functionalfragment thereof. The nucleic acid molecule may resideextra-chromosomally or will preferably integrate into the genome of theH. pylori. The term “nucleic acid” as used herein refers to a polymericform of nucleotides of any length, either ribonucleotides ordeoxynucleotides. Thus, this term includes, but is not limited to,single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. The term “heterologous nucleic acid,” asused herein, refers to a nucleic acid wherein at least one of thefollowing is true: (a) the nucleic acid is foreign (“exogenous”) to(i.e., not naturally found in) H. pylori; (b) the nucleic acid comprisestwo or more nucleotide sequences or segments that are not found in thesame relationship to each other in nature, e.g., the nucleic acid isrecombinant. “Recombinant,” as used herein, means that a particularnucleic acid (DNA or RNA) is the product of various combinations ofcloning, restriction, and/or ligation steps resulting in a constructhaving a structural coding or non-coding sequence distinguishable fromendogenous nucleic acids found in natural systems. Generally, DNAsequences encoding the structural coding sequence are assembled fromcDNA fragments and short oligonucleotide linkers, or from a series ofsynthetic oligonucleotides, to provide a synthetic nucleic acid which iscapable of being expressed from a recombinant transcriptional unitcontained in a cell or in a cell-free transcription and translationsystem. Such sequences are provided in the form of an open reading frameuninterrupted by internal non-translated sequences, or introns, whichare typically present in eukaryotic genes. Genomic DNA comprising therelevant sequences can also be used in the formation of a recombinantgene or transcriptional unit. Sequences of non-translated DNA may bepresent 5′ or 3′ from the open reading frame, where such sequences donot interfere with manipulation or expression of the coding regions, andmay indeed act to modulate production of a desired product by variousmechanisms. Thus, e.g., the term “recombinant” polynucleotide or“recombinant” nucleic acid refers to one which is not naturallyoccurring, e.g., is made by the artificial combination of two otherwiseseparated segments of sequence through human intervention. Thisartificial combination is often accomplished by either chemicalsynthesis means, or by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques. Such isusually done to replace a codon with a redundant codon encoding the sameor a conservative amino acid, while typically introducing or removing asequence recognition site. Alternatively, it is performed to jointogether nucleic acid segments of desired functions to generate adesired combination of functions. This artificial combination is oftenaccomplished by either chemical synthesis means, or by the artificialmanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques. In some embodiments, the heterologous nucleicacid sequence is introduced into a H. pylori strain of the presentinvention by a vector. By “vector” is meant a recombinant nucleic acid,generally recombinant DNA, which has been generated for the purpose ofthe expression and/or propagation of a specific nucleic acid sequence,or is to be used in the construction of other recombinant nucleic acidsequences. The vector often comprises DNA regulatory sequences as wellas the nucleic acid sequence of interest. The terms “DNA regulatorysequences”, “control elements,” and “regulatory elements,” refer totranscriptional and translational control sequences, such as promoters,enhancers, polyadenylation signals, terminators, protein degradationsignals, and the like, that provide for and/or regulate expression of anucleic acid sequence in a H. pylori cell. The term “transformation” isused interchangeably herein with “genetic modification” and refers to apermanent or transient genetic change induced in a H. pylori cellfollowing introduction of a new nucleic acid. Genetic change(“modification”) are accomplished either by incorporation of the new DNAinto the genome of the H. pylori cell, or by transient or stablemaintenance of the new DNA as an episomal element such as an expressionvector, which may contain one or more selectable markers to aid in theirmaintenance in the recombinant H. pylori cell. Suitable methods ofgenetic modification include transfection, conjugation, protoplastfusion, electroporation, particle gun technology, calcium phosphateprecipitation, direct microinjection, and the like. A general discussionof these methods are found in Ausubel, et al., Short Protocols inMolecular Biology, 3^(rd) ed., Wiley & Sons, 1995. The DNA regulatorysequences and nucleic acid sequence of interest are often “operablylinked,” which refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. For instance, a promoter is operably linked to a codingsequence if the promoter effects its transcription or expression. Asused herein, the terms “heterologous promoter” and “heterologous controlregions” refer to promoters and other control regions that are notnormally associated with a particular nucleic acid in nature. Forexample, a “transcriptional control region heterologous to a codingregion” is a transcriptional control region that is not normallyassociated with the coding region in nature. In some embodiments, thenucleic acid sequence encodes a heterologous antigen. A “heterologousantigen” is one not native to H. pylori, i.e., not expressed by H.pylori in nature or prior to introduction into H. pylori. An “antigen”refers to any immunogenic moiety or agent, generally a macromolecule,which can elicit an immunological response in a mammal. The term may beused to refer to an individual macromolecule or to a homogeneous orheterogeneous population of antigenic macromolecules. As used herein,“antigen” is generally used to refer to a protein molecule or portionthereof which contains one or more epitopes, which is encoded by anucleic acid sequences as herein defined. In various examples of theinvention, the antigen contains one or more T cell epitopes. A “T cellepitope” refers generally to those features of a peptide structure whichare capable of inducing a T cell response. In this regard, it isaccepted in the art that T cell epitopes comprise linear peptidedeterminants that assume extended conformations within thepeptide-binding cleft of MHC molecules, (Unanue et al., (1987), Science,236:551-557). As used herein, a T cell epitope is generally a peptidehaving at least about 3-5 amino acid residues, and preferably at least5-10 or more amino acid residues. The ability of a particular antigen tostimulate a cell-mediated immunological response may be determined by anumber of well-known assays, such as by lymphoproliferation (lymphocyteactivation) assays, CTL cytotoxic cell assays, or by assaying forT-lymphocytes specific for the antigen in a sensitized subject. See,e.g., Erickson et al., (1993), J. Immunol., 151:4189-4199; and Doe etal., (1994), Eur. J. Immunol., 24:2369-2376. In other examples of theinvention, the antigen contains one or more B cell epitopes. A “B cellepitope” generally refers to the site on an antigen to which a specificantibody molecule binds. The identification of epitopes which are ableto elicit an antibody response is readily accomplished using techniqueswell known in the art. See, e.g., Geysen et al., (1984), Proc. Natl.Acad. Sci. USA, 81:3998-4002 (general method of rapidly synthesisingpeptides to determine the location of immunogenic epitopes in a givenantigen); U.S. Pat. No. 4,708,871 (procedures for identifying andchemically synthesising epitopes of antigens); and Geysen et al.,(1986), Molecular Immunology, 23:709-715 (technique for identifyingpeptides with high affinity for a given antibody). In some embodiments,the nucleic acid sequence encoding one or more antigens (allergens) areinserted into a suitable H. pylori shuttle vector, e.g., a shuttleplasmid with selectable markers, e.g., antibiotic markers, to assesstheir transformability. Broadly, a suitable shuttle vector will includeone, two, three or more of the following features, a cloning site, a H.pylori origin of replication, an E. coli origin of replication, and anantibiotic resistance gene and/or selectable marker. Art-known vectorssuitable for this purpose, or readily adaptable for this purposeinclude, for example, the recombinant shuttle plasmid pHR106 describedby Roberts et al. (Appl Env Mircobiol., 54: 268-270 (1988)); the PJIR750 and PJIR 751 plasmids described by Bannam et al. (Plasmid,29:233-235 (1993)); the promoter-less PPSV promoter selection vector ofMatsushita et al. (Plasmid, 31, 317-319 (1994)); the shuttle plasmidspJIR1456 and pJIR1457, described by Lyras et al. (Plasmid, 39, 160-164(1988)); and the pAK201 shuttle vector described by Kim et al. (ApplEnviron Microbiol., 55, 360-365(1989)), the contents of which areincorporated herein by reference in their entireties. Alternatively,homologous recombination are used to introduce an exogenous sequenceinto the genome of the H. pylori. Once the vector, e.g., a shuttlevector, has been produced then nucleic acid transfer protocols are usedincluding transformation/transfection, electroporation, liposomemediated nucleic acid transfer,N-[1-(2,3-Dioloyloxy)propyl]-N,N,N-trimethyl ammonium methyl sulfatemeditated transformation, and others. One skilled in the art will bereadily able to select the appropriate tools and methods for geneticmodifications of the H. pylori according to the knowledge in the art anddesign choice. Once the H. pylori or genetically modified H. pylori ofthe present invention has been isolated, passaged through a host and/orprepared, by for example culturing it are used in the present methods.

A composition according to paragraph 8, wherein the nucleic acidmolecule resides extra-chromosomally.

A composition according to paragraph 8, wherein the nucleic acidmolecule is chromosomally inserted.

A composition according to any one of paragraphs 8 to 10, wherein theheterologous antigen or a functional fragment thereof will encode anenvironmental antigen. For example, the antigen are obtained or derivedfrom any known allergen including a recombinant allergen. Exemplaryrecombinant allergens are provided in the tabular representationprovided below:

Recombinant Allergens

Source Allergen Reference Shrimp/lobster tropomyosin Leung et al. (1996)J. Allergy Clin. Immunol. 98: 954 961 Pan s 1 Leung et al. (1998) Mol.Mar. Biol. Biotechnol. 7: 12 20 Ant Sol i 2 (venom) Schmidt et al. JAllergy Clin Immunol., 1996, 98: 82 8 Bee Phospholipase A2 (PLA) Mulleret al. J Allergy Clin Immunol, 1995, 96: 395 402 Forster et al. JAllergy Clin Immunol, 1995, 95: 1229 35 Muller et al. Clin Exp Allergy,1997, 27: 915 20 Hyaluronidase (Hya) Soldatova et al. J Allergy ClinImmunol, 1998, 101: 691 8 Cockroach Bla g Bd9OK Helm et al. J AllergyClin Immunol, 1996, 98: 172 180 Bla g 4 (a calycin) Vailes et al. JAllergy Clin Immunol, 1998. 101: 274 280 Glutathione S- Arruda et al. JBiol Chem, 1997, 272: 20907 12 transferase Per a 3 Wu et al. MolImmunol, 1997, 34: 1 8 Dust mite Der p 2 (major allergen) Lynch et al. JAllergy Clin Immunol, 1998, 101: 562 4 Hakkaart et al. Clin Exp Allergy,1998, 28: 169 74 Hakkaart et al. Clin Exp Allergy, 1998, 28: 45 52Hakkaart et al. Int Arch Allergy Immumol, 1998, 115 (2): 150 6 Muelleret al. J Biol Chem, 1997, 272: 26893 8 Der p2 variant Smith et al. JAllergy Clin Immunol, 1998, 101: 423 5 Der f2 Yasue et al. Clin ExpImmunol, 1998, 113: 1 9 Yasue et al. Cell Immunol, 1997, 181: 30 7 Derp10 Asturias et al. Biochim Biophys Acta, 1998, 1397: 27 30 Tyr p 2Eriksson et al. Eur J Biochem, 1998 Hornet Antigen 5 aka Dol m VTomalski et al. Arch Insect Biochem Physiol, 1993 (venom) 22: 303 13Mosquito Aed a 1 (salivary Xu et al. Int Arch Allergy Immunol, 1998,115: 245 51 apyrase) Yellow jacket antigen 5, hyaluronidase King et al.J Allergy Clin Immunol. 1996, 98: 558 600 and phospholipase (venom) CatFel d 1 Slunt et al J Allergy Clin Immunol, 1995, 95: 1221 8 Hoffmann etal. (1997) J Allergy Clin Immunol 99: 227 32 Hedlin Curr Opin Pediatr,1995, 7: 676 82 Cow Bos d 2 (dander; Zeiler et al. J Allergy ClinImmunol, 1997, 100: 721 7 a lipocalin) Rautiainen et al. 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Int Arch Allergy Immonol, 1995,107: 248 50 Poa pratensis Poa p9 Parronchi et al. Eur J Immunol, 1996,26: 697 703 Astwood et al. Adv Exp Med Biol, 1996, 409: 269 77 RagweedAmb a I Sun et al. Biotechnology Aug, 1995, 13: 779 86 Hirsehwehr et al.J Allergy Clin Immunol, 1998, 101: 196 206 Casale et al. J Allergy ClinImmunol, 1997, 100: 110 21 Rye Lol p I Tamborini et al. Eur J Biochem,1997, 249: 886 94 Walnut Jug r I Teuber et al. J Allergy Clin Immun.,1998, 101: 807 14 Wheat allergen Fuchs et al. J Allergy Clin Immunol,1997, 100: 356 64 Donovan et al. Electrophoresis, 1993, 14: 917 22Aspergillus Asp f 1, Asp f 2, Asp f 3, Crameri et al. Mycoses, 1998, 41Suppl 1: 56 60 Asp f 4, rAsp f 6 Hemmann et al. Eur J Immunol, 1998, 28:1155 60 Banerjee et al. J Allergy Clin Immunol, 1997, 99: 821 7 CrameriInt Arch Allergy Immunol, 1998, 115: 99 114 Crameri et al. Adv Exp MedBiol, 1996, 409: 111 6 Moser et al. 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A composition according to any one of paragraphs 8 to 11, wherein thenucleic acid molecule encoding the heterologous antigen will reside in aplasmid vector comprising (a) a nucleotide sequence encoding theheterologous antigen and (b) a control or regulatory sequenceoperatively linked thereto which is capable of controlling theexpression of the nucleic acid when the vector is transformed into a H.pylori strain.

A composition according to any one of paragraphs 1 to 12, wherein thecomposition further comprises an adjuvant. Any adjuvant known in the artmay be used.

A composition according to paragraph 13, wherein the adjuvant isselected from 5 the group consisting of alum, pertussis toxin, Lactofucopentaose III, phosphopolymer, complete Freund's adjuvant,monophosphoryl lipid A, 3-de-O-acylated monophosphoryllipid A (3D-MPL),aluminium salt, CpG-containing oligonucleotides, immunostimulatory DNAsequences, saponin, MONTANIDE® ISA 720, SAF, ISCOMS, MF-59®, SBAS-3,SBAS-4, Detox, RC-529, aminoalkyl glucosaminide 4-phosphate, 10 andLbeiF4A or combinations thereof.

A composition according to any one of paragraphs 1 to 14, wherein thecomposition is formulated to prevent or treat anergy and/or allergy in amammal. The dosage and duration of administration of the composition toa mammal will be determined by the health professional attending themammalian subject in need of treatment, and will consider the age, sexand weight of the subject, the specific H. pylori and nucleic acidmolecule being expressed or the state in which the H. pylori and/or celllysate thereof e.g., whether the H. pylori is killed or alive or thestrain of H. pylori being used. The various delivery forms of thecompositions are readily prepared for use in the practice of the presentinvention given the specific types and ratios of specific H. pylori,plasmid vectors and other delivery mechanisms described herein, andthose formulation techniques known to those in the formulary arts, suchas are described in Remington's Pharmaceutical Sciences, 20th edition,Mack Publishing Company, which text is specifically incorporated hereinby reference. One application of the composition of the invention is toalter, ameliorate, or change the immune response to one or allergens(antigens), thereby resulting in anergy. The terms “altering oraltered,” “effecting or effected” or “altering relative to” are all usedherein to imply or suggest that the specific immune response of anindividual has been modified when compared to specific immune responsebefore the methods of the invention have been used. Allergic diseasesthat are specifically considered to be prevented and/or treated by themethods of the present invention include, but are not limited to contactdermatitis (Kapsenberg et al., Immunol Today 12:392-395), chronicinflammatory disorders such as allergic atopic disorders (against commonenvironmental allergens) including allergic asthma (Walker et al.,(1992), Am. Rev. Resp. Dis. 148:109-115), atopic dermatitis (van derHeijden et al., (1991), J. Invest. Derm. 97:389-394); hyper-IgEsyndrome, Omenn's syndrome, psoriases, hay fever, allergic rhinitis,urticaria, eczema and food allergies. The H. pylori containingcomposition may be formulated for administration or delivery “orally,”“enterally,” or “non-parenterally,” i.e., by a route or mode along thealimentary canal.

A composition according to paragraph 15, wherein the allergy is selectedfrom the group consisting of contact dermatitis, chronic inflammatorydisorders, allergic atopic disorders, allergic asthma, atopicdermatitis, hyper-IgE syndrome, Omenn's syndrome, psoriases, hay feverand allergic rhinitis.

A composition according to any one of paragraphs 1 to 16, wherein thecomposition is formulated to be orally administered. Examples of “oral”routes of administration of a composition include, without limitation,swallowing liquid or solid forms of a composition from the mouth,administration of a composition through a nasojejunal or gastrostomytube, intraduodenal administration of a composition, and rectaladministration, e.g., using suppositories that release the H. pyloristrain as described herein to the lower intestinal tract of thealimentary canal.

A method of treatment or prevention of allergy in a mammal at risk ofdeveloping said method comprising the step of administering to saidmammal an effective amount of a composition comprising an isolated H.pylori cell, a cell lysate thereof or combination thereof and apharmaceutically accepted carrier, wherein said H. pylori cell is eitherkilled or incapable of colonizing the mucosa of said mammal, whereinsaid composition, upon administration, provides protective immunityagainst said allergy. Preferably, the composition is the compositionaccording to any one of paragraphs 1 to 17. The term “mucosa” in thiscontext refers to the lining of mammalian tissue including, but notlimited to oral mucosa esophageal mucosa, gastric mucosa, nasal mucosa,bronchial mucosa and uterine mucosa. Preferably, the mucosa is thegastric mucosa. Mucosal delivery may encompass delivery to the mucosa.Oral mucosal delivery includes buccal, sublingual and gingival routes ofdelivery. Accordingly, the present invention relates to a method inwhich said mucosal delivery is chosen from the group consisting ofbuccal delivery, pulmonary delivery, ocular delivery, nasal delivery andoral delivery. Preferably, said mucosal delivery is oral delivery. Theterm “mammal” or “mammalian subject” or “individual” are usedinterchangeably herein to refer to any member of the subphylum Chordata,including, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The method is intended for use in anyof the above vertebrate species. The term “treatment” is used herein tomean affecting an individual or subject, their tissue or cells to obtaina desired pharmacological and/or physiological effect, such as byprophylaxis i.e., complete or partial prevention of allergic disease orsign or symptom thereof, or by therapy i.e., partial or complete cure ofallergic disease, including: (a) preventing the allergic disease fromoccurring in a subject that may be predisposed to the allergic disease,but has not yet been diagnosed as having them; (b) inhibiting theallergic disease, i.e., arresting its development; or (c) relieving orameliorating the symptoms of the allergic disease, i.e., causeregression of the symptoms of the allergic disease.

A method of treatment or prevention of allergy in an immunologicallynaive mammal at risk of developing said allergy, said method comprisingthe step of: (i) identifying a mammal at risk of developing an allergy;(ii) administering to said mammal a composition comprising an isolatedH. pylori cell, a cell lysate thereof or combination thereof and apharmaceutically accepted carrier, wherein said H. pylori cell is eitherkilled or incapable of colonizing the mucosa of said mammal and (iii)allowing sufficient time to elapse to enable anergy to develop.Preferably, the composition is the composition according to any one ofparagraphs 1 to 17. The terms “mucosa” and “mammal” and “treatment” havethe meanings given in paragraph 18 hereof.

A method of treatment or prevention of allergy in a mammal comprisingthe step of administering to said mammal an effective amount of acomposition comprising an isolated H. pylori cell, a cell lysate thereofor combination thereof and a pharmaceutically accepted carrier, whereinsaid H. pylori cell is either killed or incapable of colonizing themucosa of said mammal, wherein said composition, upon administration,provides protective immunity against said allergy. Preferably, thecomposition is the composition according to any one of paragraphs 1 to17. The terms “mucosa” and “mammal” and “treatment” have the meaningsgiven in paragraph 18 hereof.

A method according to any one of paragraphs 18 to 20, wherein the mammalis a dog, a cat, a livestock animal, a primate or a horse.

A method according to paragraph 21, wherein the primate is a human.Adult and newborn and infant humans, are intended to be treated by thisinvention. In some embodiments, the mammal is a human child between 3months and 7 years old, not less than 6 months old, more preferably notless than 9 months old. In some embodiments, the mammal is a humanindividual older than 7 years. Because in early childhood mostindividuals will not yet have been exposed to sensitisation byenvironmental allergens, it is considered that this period provides theoptimum opportunity to predict the likely onset of allergy.

A method according to paragraph 22, wherein the human is below the ageof about 5.

A method according to paragraph 23, wherein the human is below the ageof 2 years.

A method according to any one of paragraphs 18 to 24, wherein theallergy is selected from the group consisting of contact dermatitis,chronic inflammatory disorders, allergic atopic disorders, allergicasthma, atopic dermatitis, hyper-IgE syndrome, Omenn's syndrome,psoriases, hay fever and allergic rhinitis.

A kit for treating and/or preventing allergy in a mammal comprising:

-   -   i). a composition according to any one of paragraphs 1 to 17;        and    -   ii). instructions for use in a method according to any one of        paragraphs 18 to 25.

A method of generating a H. pylori strain that is able to provideprotective immunity against allergy comprising the steps of:

-   -   (a) providing an isolated H. pylori cell that is;        -   (i) incapable of colonizing the mucosa of a mammal and/or        -   (ii) cagA minus (cagA) and optionally positive for the            toxigenic s1 and m1 alleles of the VacA gene;    -   (b) optionally passaging said H. pylori cell through an animal        host; and    -   (c) optionally inactivating or killing said H. pylori cell.

The claims defining the invention are as follows:
 1. A method ofinterrupting or slowing or arresting or preventing an atopic march orprogression of an atopic march in a human subject having or susceptibleto atopic march, the method comprising, administering to the subject aneffective amount of a composition comprising an inactivated or killed H.pylori cell or a cell lysate thereof.
 2. A method for preventing anallergic immune response to an allergen or reducing severity orincidence of an allergic immune response to an allergen in a humansubject having or susceptible to an allergic immune response to anallergen comprising administering to the subject a therapeuticallyeffective amount of killed or inactivated H. pylori cells or a celllysate thereof.
 3. The method of claim 2, wherein said method comprisesadministering a daily dosage of the H. pylori cells or cell lysatewherein each dose comprises an amount of bacteria in a rangecorresponding to between about 10⁶ cells to about 10¹² cells, or alysate thereof.
 4. The method of claim 2, wherein the H. pylori cells orcell lysate thereof is administered to a juvenile human subject toprevent eczema in the juvenile or a subsequent onset of allergy orasthma in later life.
 5. The method of claim 2, wherein the H. pyloricells or cell lysate thereof is administered over a period of at leastabout 2 weeks.
 6. The method of claim 2, wherein the H. pylori cells orcell lysate thereof is administered over a period of at least about 4weeks.
 7. The method of claim 2, wherein the H. pylori cells or celllysate thereof is administered over a period of at least about 6 weeks.8. The method of claim 2, wherein the H. pylori cells or cell lysatethereof is administered over a period of at least about 8 weeks.
 9. Themethod of claim 2, wherein the H. pylori cells or cell lysate thereof isadministered over a period of at least about 10 weeks.
 10. The method ofclaim 2, wherein the H. pylori cells or cell lysate thereof isadministered over a period of at least about 12 weeks.
 11. The method ofclaim 2, wherein said method prevents or delays an increase in a serumlevel of allergen-specific IgE antibody and/or prevents or delays anincrease in a level of one or more inflammatory cytokines inbronchioalveolar lavage (BAL) and/or prevents or delays an increase in alevel of cell infiltrate in lung in a human subject exposed to anallergen.
 12. The method of claim 2, wherein said method comprisesadministering H. pylori whole cell lysate.